Methods and devices for detection and acquisition of biomarkers

ABSTRACT

The present invention provides devices and methods for detecting and capturing molecular biomarkers from a subject in situ. Specifically, the devices contain an array of microneedles to which are attached probes specific for one or more biomarkers of interest. The devices can be used directly on a subject (e.g., via skin piercing) in detecting the biomarkers in the body of the subject (e.g., tissues, blood stream).

CROSS-REFERENCE

This application is a continuation of U.S. patent application Ser. No.15/367,015, filed Dec. 1, 2016, which is a continuation of U.S. patentapplication Ser. No. 14/106,661, filed Dec. 13, 2013 and claims thebenefit of U.S. Provisional Patent Application Ser. No. 61/737,237,filed on Dec. 14, 2012, each of which is incorporated herein byreference in its entirety.

SEQUENCE LISTING

This disclosure includes a sequence listing submitted as a text filepursuant to 37 C.P.R. § 1.52(e)(v) named 45872-701-301_SL.txt, createdon Jan. 16, 2017, with a size of 966 bytes, which is incorporated hereinby reference.

BACKGROUND

Analysis of biomarkers is fast becoming the preferred method for earlydetection of disease, patient stratification and monitoring efficacy oftreatment. Rapid and highly sensitive detection of changes in abiomarker is often technically impossible, or may require a cumbersomeprocedure involving multiple processing steps, necessitating largesample volumes and a prolonged diagnosis/prognosis timeline. The samplefrom a patient is often of a limited volume and not amenable toprocessing or to procedures requiring multiple steps that extend theprocessing time.

Current methods for detecting molecular biomarkers or biologicalanalytes of interest for diagnostic applications rely primarily onextraction of body fluid (e.g., blood, interstitial tissue fluid) from apatient. It is from this sample of fluid that specific biomarkers areassayed. More recent inventions do not directly sample clinicallyrelevant biomarkers from the site of application, but rather, requirefurther processing of body fluids. Other diagnostic methodologies thatare not based on molecular assaying, e.g., biopsies, are usuallycumbersome and fraught with misdiagnosis risks due to their inherentlyvisual and subjective nature. For localized, non-circulating biomarkers,however, this has often been the only diagnostic option to-date.

There is a need in the art for better means for detecting and analyzingbiomarkers present in the body of subjects having or at risk ofdeveloping various diseases or disorders. The present inventionaddresses this and other needs.

SUMMARY OF THE INVENTION

In one aspect, the invention provides devices for detecting orextracting one or more biomarkers from a tissue or biological sample ofa subject in situ. The devices can comprise an array of microneedles anda plurality of probes specific for the biomarkers, wherein the probesare covalently attached to the microneedles. In some of the devices, theprobes are specific for different biomarkers, and each different probeis attached to a different microneedle. In some other devices, the sameprobe for a specific biomarker is attached to two or more microneedles.The microneedles in the devices can be made of a polymer, a metal, aceramic or any other suitable material.

In some cases, the disclosure provides a device for detecting orextracting one or more biomarkers from an in situ tissue or biologicalsample of a subject, comprising one or more microneedles, and one ormore probes specific for the biomarkers, wherein the probes are attachedto the microneedles, via covalent or non-covalent linkage.

The device can comprise a first microneedle. The first microneedle canbe covalently attached to a first probe, which is specific for a firstbiomarker. Alternatively, the first microneedle can be non-covalentlyattached to the first probe. Further, the first probe can be attached toa plurality of microneedles. In some cases, the invention provides adevice comprising a first microneedle, wherein the first microneedle iscovalently or non-covalently attached to a first probe specific for afirst biomarker. In some cases, the first biomarker can be apolynucleotide. The first probe can be a polynucleotide complementary tothe first biomarker. In other cases, the first biomarker can be apolypeptide, an antibody, a metabolite, or a small molecule. Further,the first probe can be a polynucleotide, a polypeptide, a protein, anantibody, a small molecule, or a biological receptor. In some cases, thefirst needle is formed on a substrate.

The device can further comprise a second probe, which is specific for asecond biomarker. The second probe can be different from the firstprobe. The second probe can be covalently or non-covalently attached tothe first microneedle. Alternatively, the second probe can be covalentlyor non-covalently attached to a second microneedle. The second biomarkercan be a polynucleotide. The second probe can be a polynucleotidecomplementary to the second biomarker. The second biomarker can also bea polypeptide, an antibody, a metabolite, or a small molecule. Further,the second probe can be a polynucleotide, a polypeptide, a protein, anantibody, a small molecule, a biological receptor. In some cases, thefirst probe specifically binds to a first nucleotide polymorphism andthe second probe specifically binds to a second nucleotide polymorphism.In some cases, the first and second probes are different antibodies eachspecific to a different epitope of the same biomarker.

In some cases, the device provides for a microneedle comprising apolymer, a metal or a ceramic. A first probe can be attached, covalentlyor non-covalently to a plurality of microneedles. A second probe and aplurality of other probes can be attached, covalently or non-covalently,to a plurality of microneedles.

Some devices of the invention are directed to detecting nucleic acidbiomarkers. In these devices, the probes that can be conjugated to themicroneedles are oligonucleotides or polynucleotides complementary tothe biomarkers. Some of the devices employ microneedles that arefabricated with a thermoplastic polymer. The polynucleotide probes inthe devices can be conjugated to the microneedles via many suitablelinkages, including but not limited to a thiol/amino bifunctional linkeror a poly(ethylene glycol) linker. In some cases, a device of theinvention further comprises a compartment for amplifying and identifyingthe first and second biomarkers.

Some other devices of the invention are specifically designed fordetection of peptide or protein biomarkers. In some of these devices,the probes immobilized on the microneedles are antibodies specific forthe biomarkers. In various embodiments of the invention, a planarsubstrate is used to support the array of microneedles. The devices ofthe invention can additionally contain a means for amplifying thebiomarkers detected by the probes.

The device can comprise a plurality of microneedles. The plurality ofmicroneedles can comprise at least one microneedle that is covalently ornon-covalently attached to at least one probe specific for a biomarker.The biomarker can be indicative of a specific condition, including butnot limited to a skin or eye condition. In some cases, the biomarker canbe indicative of a skin condition. In some examples, the skin conditionis a skin cancer. In other cases, the biomarker can be indicative of aneye condition. In some examples, the eye condition is an eye cancer oreye inflammation.

In some cases, the disclosure provides a device comprising a pluralityof microneedles, wherein the plurality of microneedles comprises atleast one microneedle that is covalently or non-covalently attached toat least one probe specific for a biomarker and the biomarker isindicative of a skin or eye condition. In some cases, the biomarker is apolynucleotide, and the at least one probe is complementary to thebiomarker. In some cases, the at least one probe comprises differentpolynucleotide probes specific to different nucleotide polymorphisms ofthe same biomarker. In some cases, the biomarker is a peptide orpolypeptide, and the at least one probe is an antibody specific for thebiomarkers. In some cases, the at least one probe comprises differentantibodies specific to different epitopes of the same biomarker.

A device of the invention can comprise a plurality of probes specificfor a plurality of different biomarkers, wherein the plurality of probesare attached to the same or to different microneedles. In some cases, atleast two different probes are attached to the same microneedle. In somecases, the devices comprise at least two identical probes for a specificbiomarker, wherein the at least two identical probes are attached to oneor more microneedles.

Alternatively, the biomarker can also be obtained during anintraoperative procedure. The device can further comprise a sensor thatemits an optical signal when the probe detects the biomarker. In someexamples, the optical signal of the probe can change when the probedetects the biomarker.

In another aspect, the disclosure provides methods for detecting oramplifying one or more biomarkers from an in situ tissue in subject(e.g., skin, blood stream, tissue) or ex vivo tissue sample. The methodsentail (a) preparing a plurality of microneedles to which a plurality ofprobes specific for the biomarkers are covalently attached, (b)contacting the microneedles with a tissue or a biological sample of thesubject, and (c) detecting biomarkers that are bound to the probes onthe microneedles.

In some cases, the disclosure provides a method for detecting one ormore biomarkers from an in situ tissue or biological sample in asubject, comprising (a) contacting a microneedle with a tissue orbiological sample of the subject, wherein the microneedle is attached toa set of probes, and wherein the probes bind to one or more biomarkersin situ; and (b) detecting the one or more biomarkers that are bound tothe probes.

In some cases, the disclosure provides a device comprising a pluralityof microneedles, wherein the plurality of microneedles comprise at leastone microneedle that is covalently or non-covalently attached to a probespecific for a biomarker, wherein the probe comprises a sensor thatemits an optical signal when the probe detects the biomarker. In somecases, the optical signal of the probe increases when it detects thebiomarker. In some cases, the optical signal of the probe decreases whenit detects the biomarker.

In some cases, the disclosure provides a method for detecting one ormore biomarkers from an in situ tissue or biological sample in asubject, comprising (a) preparing a device comprising one or moremicroneedles to which one or more probes specific for the biomarkers arecovalently attached; (b) contacting the microneedle device with thetissue or biological sample of the subject; and (c) detecting biomarkersthat are bound to the probes on the microneedle array.

In some cases, the disclosure provides a method for detecting one ormore biomarkers from an in situ tissue or biological sample in asubject, comprising: (a) contacting a microneedle device with a tissueor biological sample of the subject, wherein the microneedle devicecomprises one or more probes specific for the one or more biomarkers,wherein at least one probe comprises a sensor that emits a visual signalwhen the probe detects the biomarkers; and (b) detecting biomarkersbased on the visual signal.

Alternatively, the method can comprise (a) contacting a microneedledevice with a tissue or biological sample of the subject, and at leasttwo probes specific for the one or more biomarker, wherein at least twoprobes specific for the biomarker are different, wherein the probes areattached to the microneedles, via covalent or non-covalent linkage, and(b) detecting biomarkers that are bound to the probes.

In some cases, the disclosure provides a method for detecting one ormore biomarkers within a tissue of a subject, comprising (a) contactinga microneedle with the tissue of the subject in situ, wherein the tissuecomprises an extracellular matrix, wherein the microneedle is covalentlyattached to a set of probes, and wherein the probes bind to a biomarkerin situ; and (b) disrupting the extracellular matrix. In some cases, theextracellular matrix is disrupted by an enzymatic activity. In somecases the disrupting the extracellular matrix comprises applyingultrasonic energy to the extracellular matrix. In some cases, theextracellular matrix is disrupted by an electric potential.

In a further example, a method can comprise (a) contacting a microneedledevice with the sample comprising an extracellular matrix, wherein themicroneedle is covalently attached to a set of probes, and wherein theprobes bind to a biomarker in situ; and (b) disrupting the extracellularmatrix. Alternatively, the method can comprise disrupting the cellularmembrane. In either example, the extracellular matrix or the cellularmembrane can be disrupted by an enzymatic activity, an ultrasonicenergy, or an electronic potential.

In yet another example, a method can comprise: (a) contacting amicroneedle device with a tissue or biological sample of the subject,wherein the microneedle device comprises one or more probes specific forthe one or more biomarkers; and (b) amplifying the biomarkers.

The biomarkers can be polynucleotides and the probes can bepolynucleotides complementary to the biomarkers. In some cases, theprobes can be different polynucleotide probes specific to differentnucleotide polymorphisms of the same biomarker

The biomarkers can be peptides or polypeptides and the probes can beantibodies specific for the biomarkers. In some cases, the probes can bedifferent antibodies specific to different epitopes of the samebiomarker.

For simultaneous detection of different biomarkers, the probes used inthe methods can be made specific for the different biomarkers. Theprobes can be attached to a different microneedle, or the probes can beattached to the same microneedle. In some other embodiments, at leasttwo microneedles can bear identical probes, or multiple probes can beused for detecting a specific biomarker.

The disclosure also provides a method of preparing a microneedle devicecomprising: (a) obtaining a solution comprising a mineral salt; (b)adding a probe and a microneedle to the solution; and (c) conjugatingthe probe to the microneedle within the solution. In some cases, themineral salt is sodium chloride. In some cases the concentration of themineral salt is less than 2.5 M.

Any suitable materials can be used in making the microneedle array usedin these methods. Examples include but are not limited to a polymer, ametal or a ceramic. Some methods of the invention are intended fordetection of nucleic acid biomarkers. In these methods, the employedprobes are oligonucleotide or polynucleotide molecules with sequencesthat are complementary to that of the biomarkers. In some of themethods, the nucleic biomarkers captured by the microneedle device aredetected and analyzed by PCR or quantitative real-time PCR (qRT-PCR). Insome of the methods, the microneedles are made of a polymer, and theprobes are attached to the microneedles via a thiol/amino bifunctionallinker. Alternatively, the microneedles are made of stainless steel andcoated with gold, and the probes are attached to the microneedles via athiol linker. In some embodiments, the microneedles can be solid. Someother methods of the invention are intended for detection of peptide orpolypeptide biomarkers. In these methods, the employed probes aremolecules that are capable of specifically binding to the biomarkers,e.g., monoclonal antibodies. Upon capturing with themicroneedle-conjugated antibodies, the peptide or protein biomarkersbound to the probes on the microneedles can be detected by addition ofan oligonucleotide-tagged secondary antibody and subsequent PCRamplification of the conjugated tag.

In some cases, the method for detecting one or more polynucleotidebiomarkers from a skin or eye tissue in a subject can comprisecontacting a microneedle device with the skin or eye tissue of thesubject. The method can further comprise contacting the microneedledevice with a dermal capillary of the subject. In other cases, themethod can comprise contacting a microneedle device with the tissue orbiological sample of the subject during an intraoperative procedure. Thetissue or biological sample can be from an organ selected from the groupconsisting of brain, heart, breast, liver, pancreas, spleen, bladder,stomach, lung, uterus, cervix, prostate, kidney, intestine, appendix,and colon. The method can further comprise contacting the microneedlesto the margins of a tumor after the tumor is removed from the subject.In some cases, a method of the disclosure detects a biomarker that ispresent in the blood of a subject.

The disclosure also provides a method for conjugating a probe to amicroneedle, wherein a mineral salt is added. Examples of mineral saltsinclude but are not limited to lithium salts, potassium salts, sodiumsalts, magnesium salts, and calcium salts, often with a halide counterion. In some cases, the mineral salt is sodium chloride. The mineralsalt can be added at a concentration between about 0.1 M to 2.0 M.Further, the mineral salt can be added at a concentration between about0.5 M to 1.5 M.

The various methods of the present invention can further comprisedetecting one or more biomarkers from a reference tissue obtained fromthe subject. Some devices or methods of the invention are designed fordetecting and acquiring biomarkers from the blood stream of a subject.In these embodiments, the probe-conjugated microneedle array iscontacted with the subject's blood stream by piercing the skin of thesubject.

In yet another aspect, the invention provides kits comprising any of thedevices for detecting or extracting biomarkers described in thisapplication. The kits can further comprise a set of reagents for apolymerase chain reaction. In some examples, the set of reagents can befor a reverse-transcriptase polymerase chain reaction. The kit canfurther comprise a holder, or written instructions for a use thereof.

In some cases, the disclosure provides a kit comprising: (a) a device,comprising a plurality of microneedles, wherein the plurality ofmicroneedles comprise at least one microneedle that is covalently ornon-covalently attached to a first specific for a biomarker; and (b) aset of reagents for a polymerase chain reaction. In some cases, the kitfurther comprises a holder. In some cases, the set of reagents comprisesa polymerase enzyme, a buffer, and a control sample. In some cases, thekit further comprises written instructions for a use thereof.

In a further aspect, the invention provides a composition comprising aplurality of microneedles coated with a substrate capable of disruptingan extracellular matrix. In some cases, the substrate can be an enzyme.The enzyme can be selected from the group consisting of serineproteases, thiol proteases, and MMPs. Specific enzymes include papain,hyaluronidase, streptokinase, streptodornase, trypsin, chymotrypsin,alpha-chymotrypsin, alpha-amlyase, DNase, collagenase, and sutilain. Inone example, the enzyme is hyaluronidase.

A further understanding of the nature and advantages of the presentinvention may be realized by reference to the remaining portions of thespecification and claims.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings (also referred to as “Figures” or “FIGs.”) ofwhich:

FIG. 1 illustrates the scheme of chemical modification of the surface ofpolycarbonate and attachment of thiol modified DNA probe.

FIG. 2 illustrates a polymer surface modified with DNA bearing 5′-Cy5modification imaged with confocal microscope using Far Red filter. Areain the left portion of the image was modified.

FIG. 3 illustrates a stainless steel surface modified with DNAcontaining fluorescent dUTP bases. Panel A. Fluorescent image of DNA onthe steel surface. Panel B. Bright field image showing the surface ofthe stainless steel sample.

FIG. 4 is a diagram of a surface of a device of the disclosure with aplurality of microneedles.

FIG. 5 illustrates a process whereby a DNA probe coupled to a goldsurface hybridizes a biomarker.

FIG. 6 illustrates a method of providing a treatment with a device ofthe disclosure.

FIG. 7 exhibits the enhanced binding of oligonucleotides to the surfaceof metal microneedles arrays upon addition of NaCl to the couplingprocedure.

FIG. 8 displays the qRT-PCR data from assaying a homogenized human skinsample using the methods of the present invention.

DETAILED DESCRIPTION

While various embodiments of the invention have been shown and describedherein, it will be obvious to those skilled in the art that suchembodiments are provided by way of example only. Numerous variations,changes, and substitutions may occur to those skilled in the art withoutdeparting from the invention. It should be understood that variousalternatives to the embodiments of the invention described herein may beemployed in practicing the invention.

The present invention provided devices and methods for detection andacquisition of biomarkers, especially molecular biomarkers, from thebody of a subject. In some embodiments, microneedle array-based deviceswith a singular or plurality of microneedles are fabricated inaccordance with the present invention. The length of individualmicroneedles on the array can vary, e.g., ranging from 50 μm to 5 mm.The devices can be fabricated by different materials, composites andmaterial combinations, including, but not limited to, metals and metalalloys, inorganic ceramics and polymers. Microneedles are chemicallymodified to couple probes for biomarkers to the microneedle surface. Thespecific coupling chemistries depend on the material of themicroneedles. Different biomarkers can be detected with cognate probesimmobilized on the same array or the same microneedle. Asexemplifications, the devices can be applied to patient on anatomicallocations such as skin, eye, tumor or other tissue for the purposes ofallowing the desired biomarkers to bind to the probes presented.Application can be done by hand (e.g., thumb pressure), an applicatordevice, with or without a strap or band to hold it in place for theduration of sampling. The physical insertion of microneedle probes candisrupt cell membranes to release genetic material, includingbiomarkers, that are available for binding to probes on the insertedmicroneedles. Extracellular biomarkers are directly available forbinding to probes, and do not need to be sampled from materials releasedfrom disrupted cells.

The time needed for biomarkers to bind to the microneedles will dependon a number of parameters, including, but not limited to, the prevalentquantity, biodistribution and concentration of biomarkers, tissueorganization, and the physical and chemical dimensions of microneedleprobes (e.g., surface area, number of probes, number of binding sites).Application time can range, e.g., from less than 10 seconds to 60minutes. Upon removal of the microneedle arrays from tissue, biomarkerscan be assayed using a number of different techniques, including, butnot limited to, PCR, quantitative PCR, protein PCR, sandwich ELISA,elution mass spectroscopy, elution Western blot, and elution ELISA.Biomarkers can be assayed either after separation from microneedles, ordirectly on the microneedles.

As illustrated in detail in the Examples, depending on the microneedlesand the probes to be coupled, various chemistries can be used in theconjugation of the probes to the microneedles. Chemical modifications ofmicroneedles made of polymer for covalent attachment of biomolecules canbe performed with numerous linkages that have been developed in the art.For example, for microneedles with a polycarbonate surface, thecarbonate monomer contains aromatic moieties that can be chemicallyderivatized post-polymerization. Treatment of needles with nitric acid,followed by reduction of the resulting nitro group gives an amine handlethrough which molecules can be coupled to the polymer backbone.Importantly, this two-step reaction can be performed on manufacturedmicroneedle arrays without compromising the integrity of the array orindividual microneedles. Using this procedure, standard amide bondcoupling reagents can be used to link a carboxylic acid-containing probeto the microneedle surface.

When the microneedles are fabricated with metal, application of the sameprinciples as described for polymer surfaces to metal surfaces isattainable. For example, stainless steel surfaces can be coated withgold via a sputter coating process, providing a chemical handle forattachment. By utilizing the well-characterized affinity of thiols forgold surfaces, one can then attach a bivalent crosslinker with a thiolon one terminus and an amine on the other to the metal surface, therebyallowing for identical coupling chemistries of the probe molecule to themicroneedle surface. Suitable methods for chemical modifications ofother types of microneedle surfaces (e.g., inorganic ceramic) forcovalent linkage of biological molecules are also known in the art.

The invention described herein has broad applicability in many differentaspects of diagnostics, including genetic (e.g., mRNA, DNA), protein,hormonal, small molecule and cellular biomarkers for diagnostics anddisease prognosis. For example, the devices or methods described hereincan be useful in detection of skin-based biomarkers, detection ofsystemically circulating biomarkers, pathogen detection (e.g., bacteria,viruses or parasites), or determination of tumor margins during surgicaltumor resection. As described herein, specific examples of skin-baseddiagnostic applications of the invention include detection of biomarkersfor cutaneous malignant melanoma, non-melanocyte skin cancers (e.g.,basal cell carcinoma, squamous cell carcinoma), autoimmune disorders(e.g., psoriasis), infectious diseases, and tropical diseases (e.g.,Buruli ulcer, onchocerciasis). Specific examples of non-skin baseddiagnostic applications of the invention include detection of markersfor oncologic diseases, hematologic diseases, cardiovascular diseases,Down's syndrome, and real-time, rapid biomarker detection duringclinical trials.

The following sections provide more detailed guidance for practicing thepresent invention.

Definitions.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which this invention pertains. The following referencesprovide one of skill with a general definition of many of the terms usedin this invention: Academic Press Dictionary of Science and Technology,Morris (Ed.), Academic Press (1^(st) ed., 1992); Oxford Dictionary ofBiochemistry and Molecular Biology, Smith et al. (Eds.), OxfordUniversity Press (revised ed., 2000); Encyclopaedic Dictionary ofChemistry, Kumar (Ed.), Anmol Publications Pvt. Ltd. (2002); Dictionaryof Microbiology and Molecular Biology, Singleton et al. (Eds.), JohnWiley & Sons (3^(rd) ed., 2002); Dictionary of Chemistry, Hunt (Ed.),Routledge (1^(st) ed., 1999); Dictionary of Pharmaceutical Medicine,Nahler (Ed.), Springer-Verlag Telos (1994); Dictionary of OrganicChemistry, Kumar and Anandand (Eds.), Anmol Publications Pvt. Ltd.(2002); and A Dictionary of Biology (Oxford Paperback Reference), Martinand Hine (Eds.), Oxford University Press (4^(th) ed., 2000). Inaddition, the following definitions are provided to assist the reader inthe practice of the invention.

Biomarkers broadly refer to any characteristics that are objectivelymeasured and evaluated as indicators of normal biological processes,pathogenic processes, or pharmacologic responses to therapeuticintervention. Unless otherwise noted, the term biomarker as used hereinspecifically refers to biomarkers that have biophysical properties,which allow their measurements in biological samples (e.g., plasma,serum, cerebrospinal fluid, bronchoalveolar lavage, biopsy). Unlessotherwise noted, the term biomarker is used interchangeably with“molecule biomarker” or “molecular markers.” Examples of biomarkersinclude nucleic acid biomarkers (e.g., oligonucleotides orpolynucleotides), peptides or protein biomarkers, lipids, andlipopolysaccharide markers.

As used herein, microneedle devices or microneedle arrays (microarrays)refer to a device comprising at least one small piercing element ormicroneedle onto which is immobilized a diagnostic agent or compound.The microneedle is capable of piercing the biological barriers in humanor other mammalian subjects (e.g., the stratum corneum of the skin) uponcontact. Preferably, the device comprises a plurality of suchmicroneedles, e.g., 2, 5, 10, 25, 50, 100, 150, 200, 250, 300, 350, 400,450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 2000, 5000,10000, 20000, or more. By conjugating diagnostic probes to themicroneedles, the microneedle devices of the invention provide a meansfor in situ detection of biomarkers in a tissue or biological sample ofa subject (e.g., blood stream or skin).

The term “polynucleotide” or “nucleic acid” as used herein refers to apolymeric form of nucleotides of any length, either ribonucleotides ordeoxyribonucleotides, that comprise purine and pyrimidine bases, orother natural, chemically or biochemically modified, non-natural, orderivatized nucleotide bases. Polynucleotides of the embodiments of theinvention include sequences of deoxyribonucleic acid (DNA), ribonucleicacid (RNA), or DNA copies of ribonucleic acid (cDNA), all of which maybe isolated from natural sources, recombinantly produced, orartificially synthesized. A further example of a polynucleotide ispolyamide polynucleotide (PNA). The polynucleotides and nucleic acidsmay exist as single-stranded or double-stranded. The backbone of thepolynucleotide can comprise sugars and phosphate groups, as maytypically be found in RNA or DNA, or modified or substituted sugar orphosphate groups. A polynucleotide may comprise modified nucleotides,such as methylated nucleotides and nucleotide analogs. The sequence ofnucleotides may be interrupted by non-nucleotide components. Thepolymers made of nucleotides such as nucleic acids, polynucleotides andpolynucleotides may also be referred to herein as nucleotide polymers.

The term “oligonucleotide” is defined as a molecule comprised of two ormore deoxyribonucleotides, preferably more than three. Its exact sizewill depend upon many factors which, in turn, depend upon the ultimatefunction and use of the oligonucleotide. The term “primer” as usedherein refers to an oligonucleotide, whether occurring naturally as in apurified restriction digest or produced synthetically, which is capableof acting as a point of initiation of synthesis when placed underconditions in which synthesis of a primer extension product, which iscomplementary to a nucleic acid strand, is induced, i.e., in thepresence of nucleotides and an inducing agent such as a DNA polymeraseand at a suitable temperature and pH. The primer may be eithersingle-stranded or double-stranded and must be sufficiently long toprime the synthesis of the desired extension product in the presence ofthe inducing agent. The exact length of the primer will depend upon manyfactors, including temperature, source of primer and the method used.For example, for diagnostic applications, depending on the complexity ofthe target sequence, the oligonucleotide primer typically contains 15-25or more nucleotides, although it may contain fewer nucleotides. Thefactors involved in determining the appropriate length of primer arereadily known to one of ordinary skill in the art.

Polypeptides are polymer chains comprised of amino acid residue monomerswhich are joined together through amide bonds (peptide bonds). The aminoacids may be the L-optical isomer or the D-optical isomer. In general,polypeptides refer to long polymers of amino acid residues, e.g., thoseconsisting of at least more than 10, 20, 50, 100, 200, 500, or moreamino acid residue monomers. A polypeptide can be a chain of at leasttwo amino acids, peptide-mimetics, a protein, a recombinant protein, anantibody (monoclonal or polyclonal), an antibody fragment, an antigen,an epitope, an enzyme, a receptor, a vitamin, or a structure analogue orcombinations thereof. However, unless otherwise noted, the termpolypeptide as used herein also encompass short peptides which typicallycontain two or more amino acid monomers, but usually not more than 10,15, or 20 amino acid monomers.

Proteins are long polymers of amino acids linked via peptide bonds andwhich may be composed of two or more polypeptide chains. Morespecifically, the term “protein” refers to a molecule composed of one ormore chains of amino acids in a specific order; for example, the orderas determined by the base sequence of nucleotides in the gene coding forthe protein. Proteins are essential for the structure, function, andregulation of the body's cells, tissues, and organs, and each proteinhas unique functions. Examples are hormones, enzymes, and antibodies. Insome embodiments, the terms polypeptide and protein may be usedinterchangeably.

“Biological sample” as used herein is a sample of biological tissue orchemical fluid that is suspected of containing a biomarker or an analyteof interest. The sample may be an ex vivo sample or in vivo sample.Samples include, for example, body fluids such as whole blood, serum,plasma, cerebrospinal fluid, urine, lymph fluids, and various externalsecretions of the respiratory, intestinal and genitourinary tracts suchas tears, saliva, semen, milk, and the like; and other biological fluidssuch as cell culture suspensions, cell extracts, cell culturesupernatants. Samples may also include tissue biopsies, e.g., from thelung, liver, brain, eye, tongue, colon, kidney, muscle, heart, breast,skin, pancreas, uterus, cervix, prostate, salivary gland, and the like.Samples may also be microbiopsies, small samples or even single cellsextracted from patients and subsequently processed, for example, usinglaser capture microdisection. A sample may be suspended or dissolved in,e.g., buffers, extractants, solvents, and the like.

As used herein, “tissue” refers to a collection of similar cells and theintracellular substances surrounding them. There are four basic tissuesin the body: 1) epithelium; 2) connective tissues, including blood,bone, and cartilage; 3) muscle tissue; and 4) nerve tissue.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another embodiment includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms another embodiment. Itwill be further understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint. The term “about” as used herein refers to a rangethat is 15% plus or minus from a stated numerical value within thecontext of the particular usage. For example, about 10 would include arange from 8.5 to 11.5.

A covalent bond is a chemical bond that involves the sharing of electronpairs between atoms. Covalent bonding includes many kinds ofinteractions, including σ-bonding, π-bonding, metal-to-metal bonding,agostic interactions, and three-center two-electron bonds. Anon-covalent interaction differs from a covalent bond in that it doesnot involve the sharing of electrons. A non-covalent bond can begenerally classified into 4 categories: electrostatic, π-effects, vander Waals forces, and hydrophobic effects.

Biomarkers.

The devices or methods described herein can be employed in variousdiagnostic applications for detecting and capturing specific biomarkers.Biomarkers can be used in clinical practice to identify risk for ordiagnose a disease, stratify patients, assess disease severity orprogression, predict prognosis, or guide treatment. In drug developmentbiomarkers may be used to help determine how a drug works in the body,to determine a biologically effective dose of a drug, to help assesswhether a drug is safe or effective, and to help identify patients mostlikely to respond to a treatment, or are least likely to suffer anadverse event when treated with a drug. Biomarkers can sometimes be usedas part of the approval process for a drug or treatment, to informregulatory decision-making.

In some cases, employing the microneedle array device described hereinand routinely practiced amplification technologies (e.g., genomics orproteomics technologies), various biomarkers in the body of a subjectcan be detected with the methods and devices of the present invention.In some cases, various biomarkers from the subject can be detected usingthe microneedle array device to capture a biomarker or set of biomarkerand then using one or more additional tagged probes to detect thecaptured biomarkers.

The biomarkers that can be detected with the present disclosure includenucleic acid-based biomarkers (DNA, RNA, mRNA transcripts, genomic DNA,tRNA, siRNA, miRNA, mitochondrial DNA, mitochondrial RNA, exosomalnucleic acids, cell-free DNA or RNA, polynucleotides carrying mutatedgenes and polymorphisms), peptides, proteins, lipids, lipidsmetabolites, and small molecules. The biomarkers include diagnosticbiomarkers (e.g., cardiac troponin for the diagnosis of myocardialinfarction), staging of disease biomarkers (e.g., brain natriureticpeptide for congestive heart failure), disease prognosis biomarkers(cancer biomarkers), and biomarkers for monitoring the clinical responseto an intervention (HbAlc for anti-diabetic treatment). They alsoinclude biomarkers used in decision making in early drug development.For instance, pharmacodynamic (PD) biomarkers are markers of a certainpharmacological response, which are of special interest in doseoptimization studies. Examples of biomarkers that have diseaseimplications include serum LDL for high cholesterol and blood pressure,P53 gene and MMPs for cancer. Additional examples of specific nucleicacid biomarkers and protein biomarkers that are suitable for detectionwith methods and devices of the present invention are described below.

Some preferred embodiments of the invention are directed to detectionand amplification of nucleic acid biomarkers. Many nucleic acidbiomarkers are known in the art. Examples include telomerase reversetranscriptase mRNA as a diagnostic biomarker for hepatocellularcarcinoma (Miura et al., Clin. Cancer Res. 11:3205-9, 2005), plasmahnRNP B1 mRNA as a biomarker of lung cancer (Sato et al., J. Cancer Res.Clin. Oncol. 134:1191-7, 2008), GD2/GM2 synthase mRNA as a biomarker forsmall cell lung cancer (Chen et al., Lung Cancer. 67:216-20, 2010),serum transforming growth factor-alpha mRNA as a prognosis biomarker forfulminant hepatitis (Miura et al., Hepatol Int. 2:213-21, 2008),Plakophilin-3 mRNA for gastrointestinal cancer (Valladares-Ayerbes etal., Cancer Epidemiol Biomarkers Prey. 19:1432-1440, 2010),metallothionein as a biomarker of heavy metal exposure (Yamada et al.,Industrial Health 39:29-32, 2001), WT1 mRNA as a biomarker formonitoring minimal residual disease in acute myeloid leukemia (Sakamotoet al., Tohoku J Exp Med. 219:169-76, 2009), and granzyme A mRNA as abiomarker for kidney transplant rejection (van Ham et al., Kidney Int'l.Aug. 18, 2010). These biomarkers, as well as various other nucleic acidbiomarkers known in the art, are all suitable for detection with methodsand devices of the present invention. Polynucleotide sequences of theseknown biomarkers were already described and characterized in the art.Based on their known sequences, probes specific for detecting thesebiomarkers (e.g., oligonucleotide primers) can be easily designed andsynthesized with routinely practiced methods of molecular biology. See,e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Press, N.Y., (3^(rd) ed., 2000); and Brent et al., CurrentProtocols in Molecular Biology, John Wiley & Sons, Inc. (ringbou ed.,2003).

In some other embodiments, peptides or protein biomarkers are detectedwith the devices and methods of the invention. Methods and devicesdescribed herein are useful for detection of various peptide or proteinbiomarkers that have been characterized in the art. Specific examples ofpeptide or protein biomarkers suitable for the invention include, butare not limited to, PSA as a biomarker of prostate cancer (Polascik etal., J. Urol. 162:293-306, 1999), cancer antigen 125 (CA125) as abiomarker for ovarian cancer (Jacobs et al., Lancet 353:1207-1210,1999), BC1, BC2 and BC3 as serum biomarkers for detection of breastcancer (Mathelin et al., Breast Cancer Res. Treat. 96:83-90, 2006),B-Defensin-2 Protein as a serum biomarker for psoriasis (Patrick et al.,PLoS ONE 4: e4725, 2009), C-reactive protein as a biomarker ofmetastasis of renal cell carcinoma (Johnson et al., Mol. Diagn. Ther.14:191-3, 2010), high molecular weight-melanoma-associated antigen as abiomarker of desmoplastic melanoma (Goto et al., Pigment Cell MelanomaRes. 23: 137-140, 2010), telomerase expression as a biomarker ofmalignant transformation in patients with inflammatory bowel disease(Gonzalo et al., Gastroenterol Hepatol. 33:288-96, 2010), andinsulin-like growth factor II mRNA-binding protein 3 (IMP3) as aprognostic biomarker for oral squamous cell carcinoma (Li et al., HeadNeck. Jul. 22, 2010). Probes for detecting these biomarkers (e.g.,monoclonal antibodies) can be readily generated with standard immunologytechniques (e.g., hybridoma technology) or obtained from commercialsuppliers (e.g., Abnova Corporation, Full Moon BioSystems and SpringBioscience).

Depending on the specific type of biomarkers to be detected, themicroneedle device described herein can be combined with various methodsknown in the art for amplifying and examining molecular entities. Manygenomic and proteomics techniques are suitable for use in the device andmethods of the invention for detecting and analyzing protein and nucleicacid markers. For example, PCR can be used for amplifying and examiningnucleic acid molecules bound to the probes on the microneedles. ELISAcan be used for analyzing peptide or protein markers captured by themicroneedle-based device of the invention. Apart from genomics andproteomics platforms biomarker assay technique, metabolomics,lipidomics, and glycomics techniques can also be used in theidentification and detection of biomarkers of other chemical classes.For example, mass spectrometry, chromatography, and nuclear magneticresonance are useful for detecting and analyzing various biologicalmolecules bound to the microneedles.

Microneedles for Attaching Diagnostic Probes.

The invention provides microneedle devices with covalently attachedmolecular probes for detection and acquisition of biomarkers from asubject in situ. The microneedle based device contains one or moremicroneedles that can be pierced into mammalian biological barriers suchas the skin or mucous membrane. Often, the microneedles arenon-invasive, or minimally-invasive. When a plurality of microneedles ispresent, the device can also have a planar substrate which supports themicroneedles. The substrate can be made of the same material as that ofthe microneedle. It can also be made of a different material. Themicroneedles employed in the invention have a length (height) that istypically in the range of 20 μm to 1 mm, preferably in the range of 50μm to 500 μm. FIG. 4 is a diagram of a surface of a device of theinvention comprising a plurality of microneedles. Each “square” in 401illustrates an individual microneedle. 401 illustrates a plurality ofmicroneedles on a surface of a device of the invention, wherein theheight of the needles ranges from about 400 μm to about 1000 μm. In someembodiments, the height of the needles is from about 20 μm to about 50μm, from about 20 μm to about 100 μm, from about 20 μm to about 150 μm,from about 20 μm to about 200 μm, from about 20 μm to about 250 μm, fromabout 20 μm to about 300 μm, from about 20 μm to about 350 μm, fromabout 20 μm to about 400 μm, from about 20 μm to about 450 μm, fromabout 20 μm to about 500 μm, from about 20 μm to about 550 μm, fromabout 20 μm to about 600 μm, from about 20 μm to about 650 μm, fromabout 20 μm to about 700 μm, from about 20 μm to about 750 μm, fromabout 20 μm to about 800 μm, from about 20 μm to about 850 μm, fromabout 20 μm to about 900 μm, from about 20 μm to about 950 μm, or fromabout 20 μm to about 1 mm. In some cases, the height of the microneedlesis less than 1 μm, less than 5 μm, less than 10 μm, less than 15 μm,less than 20 μm, less than 25 μm, less than 30 μm, less than 35 μm, lessthan 40 μm, less than 45 μm, less than 50 μm, less than 75 μm, less than100 μm, less than 150 μm, less than 200 μm, less than 250 μm, less than300 μm, less than 500 μm, less than 750 μm, less than 1000 μm, less than2000 μm, less than 3000 μm, less than 4000 μm, less than 5000 μm, lessthan 7500 μm or less than 10000 μm. In some cases, the height of themicroneedles is greater than 1 μm, greater than 5 μm, greater than 10μm, greater than 15 μm, greater than 20 μm, greater than 25 μm, greaterthan 30 μm, greater than 35 μm, greater than 40 μm, greater than 45 μm,greater than 50 μm, greater than 75 μm, greater than 100 μm, greaterthan 150 μm, greater than 200 μm, greater than 250 μm, greater than 300μm, greater than 500 μm, greater than 750 μm, greater than 1000 μm,greater than 2000 μm, greater than 3000 μm, greater than 4000 μm,greater than 5000 μm, greater than 7500 μm or greater than 10000 μm.

While the needle-shaped microneedles can be blunt-pointed objects, theyare preferably sharp-pointed objects. In some embodiments, themicroneedles have a circular cone structure with a diameter of the basegenerally in the range of 10 μm to 500 μm, preferably in the range of 20μm to 200 μm. 401 illustrates a surface of a device of the inventionwith a plurality of microneedles wherein the diameter of the base isless than 10 mm in width. For devices containing a plurality ofmicroneedles, the microneedles can be present on the devices in rows. Insome embodiments, the rows can be spaced at virtually equal intervals tothe space of the needles aligned in the row. In some embodiments, therows can be spaced at irregular intervals.

A microneedle can have a plurality of shapes, for example, a microneedlecan be round, conical, triangular, square, rectangular, pentagonal,hexagonal, heptagonal, octagonal, or any other suitable shape. Amicroneedle can be a sharp microneedle, a blunt microneedle, or anycombination thereof. For example, a device of the invention comprising aplurality of sharp microneedles can be used to penetrate the skin of asubject thereby contacting the probe(s) on the microneedle with, forexample, an RNA biomarker. A sharp microneedle can be used to disrupttissue of a biological sample, such as a layer of cells or the outermembrane of a cell. A blunt microneedle can be used to touch the surfaceof the skin of a subject thereby contacting the microneedle with, forexample, a cell-surface biomarker on the skin. In some cases, thisdisclosure provides microneedle devices that comprise microneedles withdifferent shapes (e.g., sharp and blunt microneedles).

In some embodiments, a device of the invention comprises at least 1microneedle, at least 100 microneedles, at least 200 microneedles, atleast 300 microneedles, at least 400 microneedles, at least 500microneedles, at least 600 microneedles, at least 700 microneedles, atleast 800 microneedles, at least 900 microneedles, at least 1000microneedles, at least 1100 microneedles, at least 1200 microneedles, atleast 1300 microneedles, at least 1400 microneedles, at least 1500microneedles, at least 1600 microneedles, at least 1700 microneedles, atleast 1800 microneedles, at least 1900 microneedles, at least 2000microneedles, at least 2100 microneedles, at least 2200 microneedles, atleast 2300 microneedles, at least 2400 microneedles, at least 2500microneedles, at least 2600 microneedles, at least 2700 microneedles, atleast 2800 microneedles, at least 2900 microneedles, at least 3000microneedles, at least 3100 microneedles, at least 3200 microneedles, atleast 3300 microneedles, at least 3400 microneedles, at least 3500microneedles, at least 3600 microneedles, at least 3700 microneedles, atleast 3800 microneedles, at least 3900 microneedles, at least 4000microneedles, at least 4100 microneedles, at least 4200 microneedles, atleast 4300 microneedles, at least 4400 microneedles, at least 4500microneedles, at least 4600 microneedles, at least 4700 microneedles, atleast 4800 microneedles, at least 4900 microneedles, or at least 5000microneedles.

In some embodiments, a device of the invention comprises at most 10000microneedles, at most 5000 microneedles, at most 2500 microneedles, atmost 2000 microneedles, at most 1000 microneedles, at most 500microneedles, at most 400 microneedles, at most 300 microneedles, atmost 100 microneedles, at most 90 microneedles, at most 50 microneedles,at most 40 microneedles, at most 30 microneedles, at most 20microneedles, at most 15 microneedles, at most 10 microneedles, at most9 microneedles, at most 8 microneedles, at most 7 microneedles, at most6 microneedles, at most 5 microneedles, at most 4 microneedles, at most3 microneedles, at most 2 microneedles, or 1 microneedle.

In some embodiments, a device of the invention comprises from about 1microneedle to about 100 microneedles, from about 1 microneedle to about200 microneedles, from about 1 microneedle to about 300 microneedles,from about 1 microneedle to about 400 microneedles, from about 1microneedle to about 500 microneedles, from about 1 microneedle to about600 microneedles, from about 1 microneedle to about 700 microneedles,from about 1 microneedle to about 800 microneedles, from about 1microneedle to about 900 microneedles, from about 1 microneedle to about1000 microneedles, from about 1 microneedle to about 1100 microneedles,from about 1 microneedle to about 1200 microneedles, from about 1microneedle to about 1300 microneedles, from about 1 microneedle toabout 1400 microneedles, from about 1 microneedle to about 1500microneedles, from about 1 microneedle to about 1600 microneedles, fromabout 1 microneedle to about 1700 microneedles, from about 1 microneedleto about 1800 microneedles, from about 1 microneedle to about 1900microneedles, from about 1 microneedle to about 2000 microneedles, fromabout 1 microneedle to about 2100 microneedles, from about 1 microneedleto about 2200 microneedles, from about 1 microneedle to about 2300microneedles, from about 1 microneedle to about 2400 microneedles, fromabout 1 microneedle to about 2500 microneedles, from about 1 microneedleto about 2600 microneedles, from about 1 microneedle to about 2700microneedles, from about 1 microneedle to about 2800 microneedles, fromabout 1 microneedle to about 2900 microneedles, from about 1 microneedleto about 3000 microneedles, from about 1 microneedle to about 3100microneedles, from about 1 microneedle to about 3200 microneedles, fromabout 1 microneedle to about 3300 microneedles, from about 1 microneedleto about 3400 microneedles, from about 1 microneedle to about 3500microneedles, from about 1 microneedle to about 3600 microneedles, fromabout 1 microneedle to about 3700 microneedles, from about 1 microneedleto about 3800 microneedles, from about 1 microneedle to about 3900microneedles, from about 1 microneedle to about 4000 microneedles, fromabout 1 microneedle to about 4100 microneedles, from about 1 microneedleto about 4200 microneedles, from about 1 microneedle to about 4300microneedles, from about 1 microneedle to about 4400 microneedles, fromabout 1 microneedle to about 4500 microneedles, from about 1 microneedleto about 4600 microneedles, from about 1 microneedle to about 4700microneedles, from about 1 microneedle to about 4800 microneedles, fromabout 1 microneedle to about 4900 microneedles, or from about 1microneedle to about 5000 microneedles.

The substrate and the microneedles of the arrays can be made of variousbiodegradable or non-biodegradable materials. Examples of the materialfor the microneedles or substrate include poly(methyl methacrylate),silicon, silicon dioxide, ceramic, metal (such as stainless steel,titanium, nickel, molybdenum, chromium, and cobalt), and synthetic ornatural resin material. Some embodiments use a biodegradable polymersuch as polylactic acid, polyglycolide, polylacticacid-co-polyglycolide, pullulan, capronolactone, polyurethane orpolyanhydride. In some other embodiments, a non-degradable material isemployed to fabricate the microneedle array, e.g., a polymerpolycarbonate, a synthetic or natural resin material such aspolymethacrylic acid, ethylenevinylacetate, polytetrafluoroethylene,polysulfone, or polyoxymethylene. In some embodiments, the employedmaterial comprises or is coated with a polysaccharide such as hyaluronicacid, pullulan, dextran, dextrin or chondroitin sulfate. In some cases,the microneedle is fabricated with a thermoplastic polymer.

The substrate and the microneedles of the arrays can be made of variousthermoplastic polymers. Non-limiting examples of thermoplastic polymersinclude acrylic polymers, such as poly(methyl methacrylate) (PMMA),nylon, polyethylene, polypropylene, polystyrene, polyvinyl chloride, orTeflon. In some cases, a device of the invention is fabricated with athermoplastic polymer selected from the group consisting ofpolycarbonate, poly(methyl methacrylate), polyethylene andpolypropylene.

Non-limiting examples of non-degradable polymers include, for example,silicone, hydrogels such as crosslinked poly(vinyl alcohol) andpoly(hydroxy ethylmethacrylate), ethylene-vinyl acetate, acylsubstituted cellulose acetates and alkyl derivatives thereof, partiallyand completely hydrolyzed alkylene-vinyl acetate copolymers,unplasticized polyvinyl chloride, crosslinked homo- and copolymers ofpolyvinyl acetate, crosslinked polyesters of acrylic acid and/ormethacrylic acid, polyvinyl alkyl ethers, polyvinyl fluoride,polycarbonate, polyurethane, polyamide, polysulphones, styreneacrylonitrile copolymers, crosslinked poly(ethylene oxide),poly(alkylenes), poly(vinyl imidazole), poly(esters), poly(ethyleneterephthalate), polyphosphazenes, and chlorosulphonated polyolefines,and combinations thereof. In some embodiments the polymer comprisesethylene vinyl acetate.

Non-limiting example of biodegradable polymers include polyesters suchas 3-hydroxypropionate, 3-hydroxybutyrate, 3-hydroxyvalerate,3-hydroxycaproate, 3-hydroxyheptanoate, 3-hydroxyoctanoate,3-hydroxynonanoate, 3-hydroxydecanoate, 3-hydroxyundecanoate,3-hydroxydodecanoate, 4-hydroxybutyrate, 5-hydroxyvalerate, polylactideor polylactic acid including poly(d-lactic acid), poly(l-lactic acid),poly(d,1-lactic acid), polyglycolic acid and polyglycolide,poly(lactic-co-glycolic acid), poly(lactide-co-glycolide),poly(ε-caprolactone) and polydioxanone. Polysaccharides includingstarch, glycogen, cellulose and chitin can also be used as biodegradablematerials.

402 illustrates a surface of a device of the disclosure 401 comprising aplurality of microneedles that have been coupled with at least one typeof probe. 402 can be coupled with, for example, a polynucleotide probe,a peptide probe, a protein probe, or any combination thereof. A probeattached to a microneedle on a device 402 can be covalently ornon-covalently coupled to the microneedle. Examples 1 and 2 describe infurther detail various methods for covalently coupling a probe to asurface.

A distance between the center of two microneedles on a device of thedisclosure can be calculated to determine a density of the microneedlesin the device. In some embodiments, the center-to-center distancebetween two microneedles can be less than 1000 μm, less than 900 μm,less than 800 μm, less than 700 μm, less than 600 μm, less than 500 μm,less than 400 μm, less than 300 μm, less than 200 μm, or less 100 μm. Insome embodiments, the center-to-center distance between two microneedlescan be no greater than 100 μm, no greater than 200 μm, no greater than300 μm, no greater than 400 μm, no greater than 500 μm, no greater than600 μm, no greater than 700 μm, no greater than 800 μm, no greater than900 μm, or no greater than 1000 μm.

A microneedle of the disclosure can comprise a plurality of differentdiameters or base widths. The shape of the base of a microneedle can be,for example, round, rectangular, triangular, square, pentagonal,hexagonal, heptagonal, or other geometric shape. A microneedle of thedisclosure can have a diameter or base width that is no greater than 500μm, no greater than 400 μm, no greater than 300 μm, no greater than 200μm, no greater than 100 μm, no greater than 50 μm, no greater than 40μm, no greater than 30 μm, no greater than 20 μm, no greater than 10 μm,no greater than 1000 nm, no greater than 900 nm, no greater than 800 nm,no greater than 700 nm, no greater than 600 nm, no greater than 500 nm,no greater than 400 nm, no greater than 300 nm, no greater than 200 nm,or no greater than 100 nm.

A probe can be covalently or non-covalently attached to a microneedle ata plurality of different depths within the microneedle. 403 illustratesa microneedle with a height of 600 μm and a width of 200 μm. 404illustrates a microneedle with a height of 600 μm and a width of 200 μmand a covalently attached probe at a depth of 10 μm. 405 illustrates amicroneedle with a height of 600 μm and a width of 200 μm and acovalently attached probe at a depth of 500 μm. In some cases the depthof a probe within a microneedle can be used to determine a depth in thetissue where a biomarker can be found. In some cases, a devicecomprising a plurality of microneedles with a plurality of probesattached at different depths of different microneedles can be used todetermine where in a tissue a biomarker can be found. For example, thedevice can be used to determine the size or depth of a lesion, such as acancerous lesion.

A depth of a probe can be of about 10 μm, about 20 μm, about 30 μm,about 40 μm, about 50 μm, about 60 μm, about 70 μm about 80 μm, about 90μm, about 100 μm, about 110 μm, about 120 μm, about 130 μm, about 140μm, about 150 μm, about 160 μm, about 170 μm, about 180 μm, about 190μm, about 200 μm, about 210 μm, about 220 μm, about 230 μm, about 240μm, about 250 μm, about 260 μm, about 270 μm, about 280 μm, about 290μm, about 300 μm, about 310 μm, about 320 μm, about 330 μm, about 340μm, about 350 μm, about 360 μm, about 370 μm, about 380 about 390 μm,about 400 μm, about 410 μm, about 420 μm, about 430 μm, about 440 μm,about 450 μm, about 460 μm, about 470 μm, about 480 μm, about 490 μm,about 500 μm, about 510 μm, about 520 μm, about 530 μm, about 540 μm,about 550 μm, about 560 μm, about 570 μm, about 580 μm, about 590 μm,about 600 μm about 610 μm, about 620 μm, about 630 μm, about 640 μm,about 650 μm, about 660 μm, about 670 μm, about 680 μm, about 690 μm,about 700 μm, about 710 μm, about 720 μm, about 730 μm, about 740 μm,about 750 μm, about 760 μm, about 770 μm, about 780 μm, about 790 μm,about 800 μm, about 810 μm, about 820 μm, about 830 μm, about 840 μm,about 850 μm, about 860 μm, about 870 μm, about 880 μm, about 890 μm,about 900 μm, of about 910 μm, about 920 μm, about 930 μm, about 940 μm,about 950 μm, about 960 μm, about 970 μm, about 980 μm, about 990 μm, orabout 1000 μm.

Optionally, the microneedle devices of the invention can additionallycontain an applicator unit that can be used to apply the device to asubject. The applicator unit can control various application parameters,such as the speed with which the array is applied, the force with whichthe array is applied, and/or the angle with which the array impacts atissue of the subject (e.g., the skin). In addition, the applicator mayaid in handling or otherwise transferring the array from a storage unitto the subject. In some embodiments, the applicator can be a single-use,disposable tool that serves as both a storage unit and an applicationtool. Examples of suitable applicators and methods of application ofmicroneedle arrays are disclosed in U.S. Pat. No. 6,293,925 (Safabash etal.), U.S. Pat. No. 6,743,211 (Prausnitz et al.), U.S. Pat. No.6,881,203 (Delmore et al.), and U.S. Pat. No. 6,855,131 (Trautman etal.), and United States Patent Application Publication No. 2004/0181203(Cormier et al.), United States Patent Application Publication No.2002/0032415 (Trautman et al.), and United States Patent ApplicationPublication No. 2002/0087182 (Trautman et al.). The applicator unit canhave a plurality of different shapes. In some embodiments, theapplicator unit can be, for example, linear, triangular, rectangular, ordisk-shaped. In some embodiments, the applicator unit is a penapplicator.

The microneedle arrays for conjugating diagnostic probes can be readilyproduced using materials and methods well known in the art forfabricating arrays with microprotrusion structures. See, e.g., U.S. Pat.Nos. 7,416,541, 7,332,197, 6,663,820, 6,503,231, United States PatentApplication 20100106105, and European Patent Application 2119469 A. Forexample, the microneedle array can be fabricated by wet etchingprocessing or dry etching processing using a silicon substrate,precision machining using metal or resin (such as discharge machining,laser machining, dicing processing, hot embossing, and injectionmolding), and mechanical cutting. With such processing method, theneedle part and the support part are molded into one piece. Example ofmethod of hollowing the needle part includes a method of performingsecondary processing by using laser machining and the like after theneedle part is being prepared. In some cases a microneedle of thedisclosure can be a microneedle that is solid (not hollow). In somecases a microneedle of the disclosure can be etched to increase asurface area of the microneedle.

A plurality of methods of cell disruption may be required to render thebiomarker accessible to the probe. The cells can be disrupted by eitherdisrupting the extracellular matrix, or disrupting the cell membrane. Adevice of the disclosure can cause cell disruption when, for example, atleast one microneedle contacts and perforates a biological sample, suchas a human skin.

A plurality of cells in situ or ex vivo can be disrupted with asubstance. As such, the invention further provides a compositioncomprising a plurality of microneedles coated with a substance capableof disrupting an extracellular matrix. In some cases, the substance isan enzyme. A number of enzymes, including but not limited to serineproteases, thiol proteases, and MMPs, could be useful in this process.Non-limiting examples of enzymes that can be used for cell and tissuedisruption include but are not limited to papain, hyaluronidase,streptokinase, streptodornase, trypsin, chymotrypsin,alpha-chymotrypsin, alpha-amlyase, DNase, collagenase, sutilainproteases, lysozyme, lipases, zymolase, cellulase, mutanolysin, orglycanases. In some examples, the enzyme is hyalurodinase.

Additional methods of cell and tissue disruption can include, forexample, sonication, electroporation, cryopulverization, physicaldisruption with pressure, grinding, detergent-based cell lysis, ormechanical shearing of the tissue, with for example, a homogeneizer. Forexample, the extracellular matrix or cell membrane may be disrupted byan ultrasonic energy or by an electrical potential. Application of asolvent to a biological sample may also be used to render a biomarkeravailable for hybridization with a probe.

A device of the disclosure may disrupt a biological tissue in situ or exvivo in a minimally-invasive manner. For example, a microneedle of thedisclosure can be contacted with the skin in the eye of a subject. Themicroneedle can gently disrupt the membrane of a layer of cells in theskin of the eye thereby providing access of a biomarker in the eye to aprobe(s) in the microneedle. In some embodiments, the methods anddevices of the disclosure can be applied to the identification andcharacterization of biomarkers from delicate tissues or inoperabletissues. For example biomarkers present in the eye or brain. In someembodiments, the methods and devices of the disclosure are applied tothe in situ characterization of biomarkers from a biological sample thatmay not be available for surgical removal in a biopsy, for example,certain types of brain tumors.

Probe Conjugation and Assays for Amplifying and Detecting Biomarkers.

Microneedles Conjugated with Probes.

A plurality of probes can be attached to a microneedle of thedisclosure. In some cases, the probes comprise polynucleotides (e.g.,DNA, RNA, cDNA, cRNA, etc.). Often the polynucleotide probes aredesigned to bind or hybridize a specific polynucleotide biomarker. Thisdisclosure also provides methods and devices for detecting peptide orprotein biomarkers. In these embodiments, the probes attached to themicroneedles can specifically recognize and bind to target peptides orproteins of interest. The probes can be any substance capable of bindingto a specific peptide or protein biomarker. They can be, e.g., a protein(e.g., an antibody, antigen, or fragment thereof), carbohydrate, or apolynucleotide. The polynucleotide may possess sequence specificity forthe biomarker.

The probes to be used depend on the biomarker or biomarkers to bedetected. Thus, depending the nature and number of biomarkers to bedetected, the number of probes immobilized to the microneedles can be 2,3, 4, 5, 6, 7, 8, 9, 10, or more. In some cases, the total number ofprobes in a microneedle can be from about 1 probe to about 1,000 probes,from about 1 probe to about 10,000 probes, from about 1 probe to about100,000 probes, from about 1 probe to about 1,000,000 probes, from about1 probe to about 10,000,000, from about 1 probe to about 100,000,000probes, from about 1,000 probes to about 10,000 probes, from about 1,000probes to about 100,000 probes, from about 1,000 probes to about1,000,000 probes, from about 1,000 probes to about 10,000,000, fromabout 1,000 probes to about 100,000,000 probes, from about 10,000 probesto about 100,000 probes, from about 10,000 probes to about 1,000,000probes, from about 10,000 probes to about 10,000,000, from about 10,000probes to about 100,000,000 probes, from about 100,000 probes to about1,000,000 probes, from about 100,000 probes to about 10,000,000, fromabout 100,000 probes to about 100,000,000 probes, from about 1,000,000probes to about 10,000,000, from about 1,000,000 probes to about100,000,000 probes, or from about 10,000,000 probes to about 100,000,000probes.

In some cases, the total number of probes in a microneedle is at leastabout 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8,about 9, about 10, about 12, about 14, about 16, about 18, about 20,about 25, about 30, about 35, about 40, about 45, about 50, about 60,about 70, about 80, about 90, about 100, about 150, about 200, about250, about 300, about 350, about 400, about 450, about 500, about 600,about 700, about 800, about 900, about 1000, about 1100, about 1200,about 1300, about 1400, about 1500, about 2000, about 2500, about 3000,about 3500, about 4000, about 4500, about 5000, about 6000, about 7000,about 8000, about 9000, about 10000, about 12000, about 14000, about16000, about 18000, about 20000, about 30000, about 40000, about 50000,about 60000, about 70000, about 80000, about 90000, about 100000, about200000, about 300000, about 400000, about 500000, about 600000, about700000, about 800000, about 900000, about 1,000,000, about 2,000,000,about 3,000,000, about 4,000,000, about 5,000,000, about 6,000,000,about 7,000,000, about 8,000,000, about 9,000,000, about 10,000,000,about 20,000,000, about 30,000,000, about 40,000,000, about 50,000,000,about 60,000,000, about 70,000,000, about 80,000,000, about 90,000,000,or about 100,000,000 probes.

In some cases, the total number of probes in a microneedle is less thanabout 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8,about 9, about 10, about 12, about 14, about 16, about 18, about 20,about 25, about 30, about 35, about 40, about 45, about 50, about 60,about 70, about 80, about 90, about 100, about 150, about 200, about250, about 300, about 350, about 400, about 450, about 500, about 600,about 700, about 800, about 900, about 1000, about 1100, about 1200,about 1300, about 1400, about 1500, about 2000, about 2500, about 3000,about 3500, about 4000, about 4500, about 5000, about 6000, about 7000,about 8000, about 9000, about 10000, about 12000, about 14000, about16000, about 18000, about 20000, about 30000, about 40000, about 50000,about 60000, about 70000, about 80000, about 90000, about 100000, about200000, about 300000, about 400000, about 500000, about 600000, about700000, about 800000, about 900000, about 1,000,000, about 2,000,000,about 3,000,000, about 4,000,000, about 5,000,000, about 6,000,000,about 7,000,000, about 8,000,000, about 9,000,000, about 10,000,000,about 20,000,000, about 30,000,000, about 40,000,000, about 50,000,000,about 60,000,000, about 70,000,000, about 80,000,000, about 90,000,000,or about 100,000,000 probes.

In addition, a probe for the biomarker can be immobilized to multiplemicroneedles in the device of the disclosure, particularly for detectionof a biomarker of low concentration, as described further herein. Insome cases, a single microneedle comprises multiple different probescapable of binding or detecting the same biomarker. In some cases, asingle microneedle comprises at least 2 different probes for the samebiomarker, at least 3 different probes, at least 4 different probes, atleast 5 different probes, at least 6 different probes, at least 7different probes, at least 8 different probes, at least 9 differentprobes, at least 10 different probes, at least 11 different probes, atleast 12 different probes, at least 13 different probes, at least 14different probes, at least 15 different probes, at least 16 differentprobes, at least 17 different probes, at least 18 different probes, atleast 19 different probes, at least 20 different probes, at least 21different probes, at least 22 different probes, at least 23 differentprobes, at least 24 different probes, at least 25 different probes, atleast 26 different probes, at least 27 different probes, at least 28different probes, at least 29 different probes, at least 30 differentprobes, at least 40 different probes, at least 41 different probes, atleast 42 different probes, at least 43 different probes, at least 44different probes, at least 45 different probes, at least 46 differentprobes, at least 47 different probes, at least 48 different probes, atleast 49 different probes, or at least 50 different probes for the samebiomarker. In some cases, the same microneedle comprises more than 50different types of probes for the same biomarker.

In some cases, the same microneedle comprises a plurality of differentprobes. The different probes can be specific for the same biomarker orfor a different biomarker. A microneedle can comprise at least 2different probes, at least 10 different probes, at least 100 differentprobes, at least 200 different probes, at least 300 different probes, atleast 400 different probes, at least 500 different probes, at least 600different probes, at least 700 different probes, at least 800 differentprobes, at least 900 different probes, at least 1,000 different probes,at least 1,100 different probes, at least 1,200 different probes, atleast 1,300 different probes, at least 1,400 different probes, at least1,500 different probes, at least 1,600 different probes, at least 1,700different probes, at least 1,800 different probes, at least 1,900different probes, at least 2,000 different probes, at least 2,100different probes, at least 2,200 different probes, at least 2,300different probes, at least 2,400 different probes, at least 2,500different probes, at least 2,600 different probes, at least 2,700different probes, at least 2,800 different probes, at least 2,900different probes, at least 3,000 different probes, at least 3,100different probes, at least 3,200 different probes, at least 3,300different probes, at least 3,400 different probes, at least 3,500different probes, at least 3,600 different probes, at least 3,700different probes, at least 3,800 different probes, at least 3,900different probes, at least 4,000 different probes, at least 4,100different probes, at least 4,200 different probes, at least 4,300different probes, at least 4,400 different probes, at least 4,500different probes, at least 4,600 different probes, at least 4,700different probes, at least 4,800 different probes, at least 4,900different probes, at least 5,000 different probes, at least 5,100different probes, at least 5,200 different probes, at least 5,300different probes, at least 5,400 different probes, at least 5,500different probes, at least 5,600 different probes, at least 5,700different probes, at least 5,800 different probes, at least 5,900different probes, at least 6,000 different probes, at least 6,100different probes, at least 6,200 different probes, at least 6,300different probes, at least 6,400 different probes, at least 6,500different probes, at least 6,600 different probes, at least 6,700different probes, at least 6,800 different probes, at least 6,900different probes, at least 7,000 different probes, at least 7,100different probes, at least 7,200 different probes, at least 7,300different probes, at least 7,400 different probes, at least 7,500different probes, at least 7,600 different probes, at least 7,700different probes, at least 7,800 different probes, at least 7,900different probes, at least 8,000 different probes, at least 8,100different probes, at least 8,200 different probes, at least 8,300different probes, at least 8,400 different probes, at least 8,500different probes, at least 8,600 different probes, at least 8,700different probes, at least 8,800 different probes, at least 8,900different probes, at least 9,000 different probes, at least 9,100different probes, at least 9,200 different probes, at least 9,300different probes, at least 9,400 different probes, at least 9,500different probes, at least 9,600 different probes, at least 9,700different probes, at least 9,800 different probes, at least 9,900different probes, or at least 10,000 different probes. A microneedle cancomprise less than 2 different probes, less than 10 different probes,less than 100 different probes, less than 200 different probes, lessthan 300 different probes, less than 400 different probes, less than 500different probes, less than 600 different probes, less than 700different probes, less than 800 different probes, less than 900different probes, less than 1,000 different probes, less than 1,100different probes, less than 1,200 different probes, less than 1,300different probes, less than 1,400 different probes, less than 1,500different probes, less than 1,600 different probes, less than 1,700different probes, less than 1,800 different probes, less than 1,900different probes, less than 2,000 different probes, less than 2,100different probes, less than 2,200 different probes, less than 2,300different probes, less than 2,400 different probes, less than 2,500different probes, less than 2,600 different probes, less than 2,700different probes, less than 2,800 different probes, less than 2,900different probes, less than 3,000 different probes, less than 3,100different probes, less than 3,200 different probes, less than 3,300different probes, less than 3,400 different probes, less than 3,500different probes, less than 3,600 different probes, less than 3,700different probes, less than 3,800 different probes, less than 3,900different probes, less than 4,000 different probes, less than 4,100different probes, less than 4,200 different probes, less than 4,300different probes, less than 4,400 different probes, less than 4,500different probes, less than 4,600 different probes, less than 4,700different probes, less than 4,800 different probes, less than 4,900different probes, less than 5,000 different probes, less than 5,100different probes, less than 5,200 different probes, less than 5,300different probes, less than 5,400 different probes, less than 5,500different probes, less than 5,600 different probes, less than 5,700different probes, less than 5,800 different probes, less than 5,900different probes, less than 6,000 different probes, less than 6,100different probes, less than 6,200 different probes, less than 6,300different probes, less than 6,400 different probes, less than 6,500different probes, less than 6,600 different probes, less than 6,700different probes, less than 6,800 different probes, less than 6,900different probes, less than 7,000 different probes, less than 7,100different probes, less than 7,200 different probes, less than 7,300different probes, less than 7,400 different probes, less than 7,500different probes, less than 7,600 different probes, less than 7,700different probes, less than 7,800 different probes, less than 7,900different probes, less than 8,000 different probes, less than 8,100different probes, less than 8,200 different probes, less than 8,300different probes, less than 8,400 different probes, less than 8,500different probes, less than 8,600 different probes, less than 8,700different probes, less than 8,800 different probes, less than 8,900different probes, less than 9,000 different probes, less than 9,100different probes, less than 9,200 different probes, less than 9,300different probes, less than 9,400 different probes, less than 9,500different probes, less than 9,600 different probes, less than 9,700different probes, less than 9,800 different probes, less than 9,900different probes, or less than 10,000 different probes.

In some cases, the plurality of probes are identical (e.g., identicalcopies of the same polynucleotide or antibody). In some embodiments, amicroneedle can be associated with numerous copies of the same probe(e.g., greater than 2, 5, 10, 50, 100, 1000, 5000, 7500, 10000, or 50000copies of the same probe). For example, a microneedle can comprise aplurality of copies of a polynucleotide probe designed to hybridize thesame polymorphism or biomarker. In some cases, a microneedle maycomprise a plurality of copies of an antibody probe designed to bind thesame epitope.

In some cases, a microneedle comprises polynucleotide probes. The probesmay be designed to detect different biomarkers associated with the samedisease, disorder or condition. In some cases, a first probe recognizesa polymorphism (e.g., DNA polymorphism, RNA polymorphism) associatedwith a disease and a second probe recognizes a different polymorphismassociated with the same disease. For example, a first DNA probe on amicroneedle can be designed to detect a first polymorphism of an RNAbiomarker associated with onchocerciasis, a skin condition. A second DNAprobe on a microneedle can be designed to detect a second polymorphismof an RNA biomarker associated with onchocerciasis. A polymorphism canbe, for example, a single nucleotide polymorphism (SNP). Genetic andgenomic variations can comprise a single SNP or a plurality of SNPs.SNPs can occur at a single locus, or at many loci. Individuals who carrya particular SNP allele at one locus can predictably carry specific SNPalleles at other loci. A correlation of SNPs can provide an associationbetween alleles predisposing an individual to disease or condition. Insome cases, the different polynucleotide probes are designed to detectdifferent biomarkers associated with different conditions. For example,one probe may detect a biomarker of a disease, while the other probe maydetect a housekeeping gene or gene product. In some cases, themicroneedles are attached to polynucleotides, polypeptides, or a mixtureof polynucleotides and polypeptides.

A microneedle can also be associated with a plurality of differentprotein or antibody probes. For example, a first antibody probe on amicroneedle can be designed to detect a first epitope of an antigenassociated with, for example, a skin cancer. A second antibody probe canbe designed to detect a second epitope associated with the antigen. Or,in some cases, the second antibody probe can detect an epitopeassociated with a different skin condition.

In some cases, this disclosure provides a microneedle device comprisinga set of microneedles, wherein each microneedle in the set comprisesidentical probes or set of probes. In some embodiments, an identicalprobe is attached to a plurality of microneedles of a device. Anidentical probe can be attached to, for example, about 1% of themicroneedles, about 5% of the microneedles, about 10% of themicroneedles, about 15% of the microneedles, about 20% of themicroneedles, about 25% of the microneedles, about 30% of themicroneedles, about 35% of the microneedles, about 40% of themicroneedles, about 45% of the microneedles, about 50% of themicroneedles, about 55% of the microneedles, about 60% of themicroneedles, about 65% of the microneedles, about 70% of themicroneedles, about 75% of the microneedles, about 80% of themicroneedles, about 85% of the microneedles, about 90% of themicroneedles, about 95% of the microneedles, or about 100% of themicroneedles. In some embodiments, an identical probe is attached to nogreater than 5% of the microneedles, no greater than 10% of themicroneedles, no greater than 15% of the microneedles, no greater than20% of the microneedles, no greater than 25% of the microneedles, nogreater than 30% of the microneedles, no greater than 35% of themicroneedles, no greater than 40% of the microneedles, no greater than45% of the microneedles, no greater than 50% of the microneedles, nogreater than 55% of the microneedles, no greater than 60% of themicroneedles, no greater than 70% of the microneedles, no greater than75% of the microneedles, no greater than 80% of the microneedles, nogreater than 85% of the microneedles, no greater than 90% of themicroneedles, no greater than 95% of the microneedles, or no greaterthan 99% of the microneedles.

In some cases, a set of microneedles may comprise at least onemicroneedle attached to a first probe and at least one microneedleattached to a second probe that is different from the first probe. Forexample, as described herein, the first probe may be a polynucleotide orpolypeptide (e.g., antibody, protein) that specifically binds abiomarker of a disease or disorder and the second probe may be apolynucleotide or polypeptide that specifically binds a differentbiomarker associated with the same disease or disorder. In some cases,the first probe may be a polynucleotide or polypeptide (e.g., antibody,protein) that specifically binds a biomarker of a disease or disorderand the second probe may be a polynucleotide or polypeptide thatspecifically binds a different biomarker associated with a differentdisease, disorder, or condition. In some cases, the different disease,condition, or disorder is associated with the same organ. For example,the first probe may be associated with a first disease, disorder, orcondition associated with skin; and the second probe may be associatedwith a second disease, disorder, or condition associated with skin oreye. In some cases, the device may comprise an array of microneedles,wherein each microneedle comprises a probe that detects a biomarkerassociated with a different disease, disorder, or condition associatedwith the same organ. The array of microneedles may comprise greater than2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 75, 100, 150, 200, 50, or1000 microneedles associated with different diseases, disorders, orconditions. In some cases, the different diseases, disorders, orconditions are associated with different organs (e.g., greater than 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 organs).

In some cases, the microneedle device comprises a plurality of arrays ofmicroneedles; often, the plurality of arrays of microneedles aresuitable for multiplexed reactions. In some cases, the plurality ofarrays comprises two or more arrays of microneedles, wherein the arraysare designed to detect a different biomarker. In some cases, a firstarray of microneedles may be designed to detect a biomarker associatedwith disease, disorder, or condition, and the second array ofmicroneedles is designed to detect a different biomarker associated withthe same disease, disorder, or condition. In some cases, a first arrayof microneedles may be designed to detect a biomarker associated withdisease, disorder, or condition, and a second array of microneedles maybe designed to detect a different biomarker associated with a differentdisease, disorder, or condition. In some cases, a first array ofmicroneedles may be designed to detect a plurality of biomarkersassociated with a disease, disorder, or condition; and a second array ofmicroneedles may be designed to detect a plurality of biomarkersassociated with a different disease, disorder, or condition. In somecases, the second array of microneedles may be designed to detectcontrol biomarkers (e.g., housekeeping genes), either positive controlsor negative controls.

In some cases, the methods and devices provided herein can be used toperform multiplex reactions with or without using fluorescence. Forexample, each microneedle can be inserted into its own cavity (e.g.,pinprick cavity), containing unique PCR reagents (e.g., unique probes orprimers). PCR reactions can be conducted and the samples can be analyzedfor various biomarkers. In some cases, the multiplex reactions areperformed with fluorescently-labeled probes, or probes that emit adifferent optical signal. In some cases, no fluorescence is used.

A microneedle device described herein may comprise any number of probes;often, the probes are attached to multiple needles within the device.The probes may be identical or different. In addition, a probe for thebiomarker can be immobilized to multiple microneedles in the device ofthe disclosure, particularly for detection of a biomarker of lowconcentration. In some cases, the total number of probes in amicroneedle device can be from about 1 probe to about 1,000 probes, fromabout 1 probe to about 10,000 probes, from about 1 probe to about100,000 probes, from about 1 probe to about 1,000,000 probes, from about1 probe to about 10,000,000, from about 1 probe to about 100,000,000probes, from about 1,000 probes to about 10,000 probes, from about 1,000probes to about 100,000 probes, from about 1,000 probes to about1,000,000 probes, from about 1,000 probes to about 10,000,000, fromabout 1,000 probes to about 100,000,000 probes, from about 10,000 probesto about 100,000 probes, from about 10,000 probes to about 1,000,000probes, from about 10,000 probes to about 10,000,000, from about 10,000probes to about 100,000,000 probes, from about 100,000 probes to about1,000,000 probes, from about 100,000 probes to about 10,000,000, fromabout 100,000 probes to about 100,000,000 probes, from about 1,000,000probes to about 10,000,000, from about 1,000,000 probes to about100,000,000 probes, or from about 10,000,000 probes to about 100,000,000probes.

In some cases, the total number of probes in a microneedle device is atleast about 1, about 2, about 3, about 4, about 5, about 6, about 7,about 8, about 9, about 10, about 12, about 14, about 16, about 18,about 20, about 25, about 30, about 35, about 40, about 45, about 50,about 60, about 70, about 80, about 90, about 100, about 150, about 200,about 250, about 300, about 350, about 400, about 450, about 500, about600, about 700, about 800, about 900, about 1000, about 1100, about1200, about 1300, about 1400, about 1500, about 2000, about 2500, about3000, about 3500, about 4000, about 4500, about 5000, about 6000, about7000, about 8000, about 9000, about 10000, about 12000, about 14000,about 16000, about 18000, about 20000, about 30000, about 40000, about50000, about 60000, about 70000, about 80000, about 90000, about 100000,about 200000, about 300000, about 400000, about 500000, about 600000,about 700000, about 800000, about 900000, about 1,000,000, about2,000,000, about 3,000,000, about 4,000,000, about 5,000,000, about6,000,000, about 7,000,000, about 8,000,000, about 9,000,000, about10,000,000, about 20,000,000, about 30,000,000, about 40,000,000, about50,000,000, about 60,000,000, about 70,000,000, about 80,000,000, about90,000,000, or about 100,000,000 probes.

In some cases, the total number of probes in a microneedle device isless than about 1, about 2, about 3, about 4, about 5, about 6, about 7,about 8, about 9, about 10, about 12, about 14, about 16, about 18,about 20, about 25, about 30, about 35, about 40, about 45, about 50,about 60, about 70, about 80, about 90, about 100, about 150, about 200,about 250, about 300, about 350, about 400, about 450, about 500, about600, about 700, about 800, about 900, about 1000, about 1100, about1200, about 1300, about 1400, about 1500, about 2000, about 2500, about3000, about 3500, about 4000, about 4500, about 5000, about 6000, about7000, about 8000, about 9000, about 10000, about 12000, about 14000,about 16000, about 18000, about 20000, about 30000, about 40000, about50000, about 60000, about 70000, about 80000, about 90000, about 100000,about 200000, about 300000, about 400000, about 500000, about 600000,about 700000, about 800000, about 900000, about 1,000,000, about2,000,000, about 3,000,000, about 4,000,000, about 5,000,000, about6,000,000, about 7,000,000, about 8,000,000, about 9,000,000, about10,000,000, about 20,000,000, about 30,000,000, about 40,000,000, about50,000,000, about 60,000,000, about 70,000,000, about 80,000,000, about90,000,000, or about 100,000,000 probes.

In some cases, a microneedle device comprises at least 2 differentprobes for the same biomarker, at least 3 different probes, at least 4different probes, at least 5 different probes, at least 6 differentprobes, at least 7 different probes, at least 8 different probes, atleast 9 different probes, at least 10 different probes, at least 11different probes, at least 12 different probes, at least 13 differentprobes, at least 14 different probes, at least 15 different probes, atleast 16 different probes, at least 17 different probes, at least 18different probes, at least 19 different probes, at least 20 differentprobes, at least 21 different probes, at least 22 different probes, atleast 23 different probes, at least 24 different probes, at least 25different probes, at least 26 different probes, at least 27 differentprobes, at least 28 different probes, at least 29 different probes, atleast 30 different probes, at least 40 different probes, at least 41different probes, at least 42 different probes, at least 43 differentprobes, at least 44 different probes, at least 45 different probes, atleast 46 different probes, at least 47 different probes, at least 48different probes, at least 49 different probes, or at 50 different leastprobes for the same biomarker. In some cases, the same microneedlecomprises more than 50 different types of probes for the same biomarker.

In some cases, a microneedle device comprises a plurality of differentprobes. The different probes can be specific for the same biomarker orfor a different biomarker. A microneedle device can comprise at least 2different probes, at least 10 different probes, at least 100 differentprobes, at least 200 different probes, at least 300 different probes, atleast 400 different probes, at least 500 different probes, at least 600different probes, at least 700 different probes, at least 800 differentprobes, at least 900 different probes, at least 1,000 different probes,at least 1,100 different probes, at least 1,200 different probes, atleast 1,300 different probes, at least 1,400 different probes, at least1,500 different probes, at least 1,600 different probes, at least 1,700different probes, at least 1,800 different probes, at least 1,900different probes, at least 2,000 different probes, at least 2,100different probes, at least 2,200 different probes, at least 2,300different probes, at least 2,400 different probes, at least 2,500different probes, at least 2,600 different probes, at least 2,700different probes, at least 2,800 different probes, at least 2,900different probes, at least 3,000 different probes, at least 3,100different probes, at least 3,200 different probes, at least 3,300different probes, at least 3,400 different probes, at least 3,500different probes, at least 3,600 different probes, at least 3,700different probes, at least 3,800 different probes, at least 3,900different probes, at least 4,000 different probes, at least 4,100different probes, at least 4,200 different probes, at least 4,300different probes, at least 4,400 different probes, at least 4,500different probes, at least 4,600 different probes, at least 4,700different probes, at least 4,800 different probes, at least 4,900different probes, at least 5,000 different probes, at least 5,100different probes, at least 5,200 different probes, at least 5,300different probes, at least 5,400 different probes, at least 5,500different probes, at least 5,600 different probes, at least 5,700different probes, at least 5,800 different probes, at least 5,900different probes, at least 6,000 different probes, at least 6,100different probes, at least 6,200 different probes, at least 6,300different probes, at least 6,400 different probes, at least 6,500different probes, at least 6,600 different probes, at least 6,700different probes, at least 6,800 different probes, at least 6,900different probes, at least 7,000 different probes, at least 7,100different probes, at least 7,200 different probes, at least 7,300different probes, at least 7,400 different probes, at least 7,500different probes, at least 7,600 different probes, at least 7,700different probes, at least 7,800 different probes, at least 7,900different probes, at least 8,000 different probes, at least 8,100different probes, at least 8,200 different probes, at least 8,300different probes, at least 8,400 different probes, at least 8,500different probes, at least 8,600 different probes, at least 8,700different probes, at least 8,800 different probes, at least 8,900different probes, at least 9,000 different probes, at least 9,100different probes, at least 9,200 different probes, at least 9,300different probes, at least 9,400 different probes, at least 9,500different probes, at least 9,600 different probes, at least 9,700different probes, at least 9,800 different probes, at least 9,900different probes, or at least 10,000 different probes. In some cases, amicroneedle device can comprise less than 2 different probes, less than10 different probes, less than 100 different probes, less than 200different probes, less than 300 different probes, less than 400different probes, less than 500 different probes, less than 600different probes, less than 700 different probes, less than 800different probes, less than 900 different probes, less than 1,000different probes, less than 1,100 different probes, less than 1,200different probes, less than 1,300 different probes, less than 1,400different probes, less than 1,500 different probes, less than 1,600different probes, less than 1,700 different probes, less than 1,800different probes, less than 1,900 different probes, less than 2,000different probes, less than 2,100 different probes, less than 2,200different probes, less than 2,300 different probes, less than 2,400different probes, less than 2,500 different probes, less than 2,600different probes, less than 2,700 different probes, less than 2,800different probes, less than 2,900 different probes, less than 3,000different probes, less than 3,100 different probes, less than 3,200different probes, less than 3,300 different probes, less than 3,400different probes, less than 3,500 different probes, less than 3,600different probes, less than 3,700 different probes, less than 3,800different probes, less than 3,900 different probes, less than 4,000different probes, less than 4,100 different probes, less than 4,200different probes, less than 4,300 different probes, less than 4,400different probes, less than 4,500 different probes, less than 4,600different probes, less than 4,700 different probes, less than 4,800different probes, less than 4,900 different probes, less than 5,000different probes, less than 5,100 different probes, less than 5,200different probes, less than 5,300 different probes, less than 5,400different probes, less than 5,500 different probes, less than 5,600different probes, less than 5,700 different probes, less than 5,800different probes, less than 5,900 different probes, less than 6,000different probes, less than 6,100 different probes, less than 6,200different probes, less than 6,300 different probes, less than 6,400different probes, less than 6,500 different probes, less than 6,600different probes, less than 6,700 different probes, less than 6,800different probes, less than 6,900 different probes, less than 7,000different probes, less than 7,100 different probes, less than 7,200different probes, less than 7,300 different probes, less than 7,400different probes, less than 7,500 different probes, less than 7,600different probes, less than 7,700 different probes, less than 7,800different probes, less than 7,900 different probes, less than 8,000different probes, less than 8,100 different probes, less than 8,200different probes, less than 8,300 different probes, less than 8,400different probes, less than 8,500 different probes, less than 8,600different probes, less than 8,700 different probes, less than 8,800different probes, less than 8,900 different probes, less than 9,000different probes, less than 9,100 different probes, less than 9,200different probes, less than 9,300 different probes, less than 9,400different probes, less than 9,500 different probes, less than 9,600different probes, less than 9,700 different probes, less than 9,800different probes, less than 9,900 different probes, or less than 10,000different probes.

Probes for detecting various biological markers can be obtainedcommercially or synthesized in accordance with methods well known in theart. The probes can be designed according to any suitable methods. Forexample, computerized search program can be used to design nucleic acidprobes specific for target biomarker sequences (e.g., mRNAs) withminimal cross hybridization and similar hybridization efficiency. Suchexemplary programs include Oligo 5.0 (National Biosciences Inc.), Primer3 (MIT), and Array Designer (Telechem International Inc.). Thenucleotide probes used in the present methods can have any suitablelength, e.g., from about 15 to about 100 nucleotides. For proteinbiomarkers, specific probes (antibodies) for detecting the biomarkerscan also be readily generated or commercially obtained. The nucleotideprobes or polypeptide probes used in the present methods can comprise adetectable label. Any suitable label can be used. For example, thedetectable label can be detected by optical, magnetic, mechanic,spectroscopic, photochemical, biochemical, immunochemical, radioactiveor enzymatic means. In some embodiments, the detectable label is afluorescent or chemiluminescent label, such as GFP; a magnetic moiety; aprotein, such as avidin, streptavidin; or a peptide tag, such as ahistidine tag or a FLAG tag.

Immobilized probes are present on the surface of the microneedle devicesdescribed herein. The immobilized probes may bind covalently ornoncovalently to the surface of the microneedles by methods known in theart or the specific linking methods described in the Examples herein.For example, the probes can be conjugated to the microneedles via, e.g.,a biotin-avidin or biotin-streptavidin interaction, a Protein Ainteraction, a Protein G interaction, a goat anti-mouse Fc interaction,an amide bond, or through any other covalent or noncovalent interaction.The probes can be covalently attached to the microneedles with orwithout a suitable spacer element between probe and microneedle surfacesuch as poly(ethylene glycol) (PEG). Methods routinely practiced in theart for immobilizing antibody probes or nucleotide probes can be readilyemployed and modified as appropriate in the practice of the invention.Such methods are described in the art, e.g., Mendoza et al.,Biotechniques 27:778-786, 1999; Arenkov et al., Anal. Biochem.278:123-131, 2000; Zhu et al., Nat. Genet. 26:283-289, 2000; MacBeath etal., Science 289:1760-1763, 2000; Jyoung et al., Biosens. Bioelectron.21:2315-2319, 2006; Lu et al., Anal. Chem. 67:83 87, 1995; Vijayendranet al., Anal. Chem. 73:471-480, 2001; Nakanishi et al., Anal. Chem. 68:1695-1700, 1996; Rowe et al., Anal. Chem. 71:433-439, 1999; Day et al.,Bichem. J. 278:735-740, 1991; Fodor et al., Science 251: 767-773, 1991;Schene et al., Science 270: 467-470, 1995; Lamture et al., Nucl. AcidsRes. 22: 2121-2125, 1994; Guo et al., Nucl. Acids Res. 22: 5456-5465,1994; and PCT publications WO00/22108, WO01/75447 and WO02/12891.

The probes can also be modified with a reactive moiety and attached tothe gold coated surface of one or more microneedles. The reactive moietycan be a thiol group. In some cases, a mineral salt can be added.Examples of mineral salts include but are not limited to lithium salts,potassium salts, sodium salts, magnesium salts, and calcium salts, oftenwith a halide counter ion. In some cases, the mineral salt is sodiumchloride. The mineral salt may be present at a concentration preferablybetween about 0.1 M to 2.0 M, about 0.2 M to 2.0 M, about 0.2 M to 1.5 Mor about 0.5 M to 1.5M. In some cases, the concentration of the mineralsalt is less than about 0.1 M, less than about 0.2 M, less than about0.2 M, less than about 0.3 M, less than about 0.4 M, less than about 0.5M, less than about 1.0 M, less than about 1.1 M, less than about 1.2 M,less than about 1.3 M, less than about 1.4 M, less than about 1.5 M,less than about 1.6 M, less than about 1.7 M, less than about 1.8 M,less than about 1.9 M, less than about 2.0 M, less than about 2.5 M,less than about 3.0 M, less than about 3.5 M, less than about 4.0 M, orless than about 4.5 M. In some cases, the concentration of the mineralsalt is greater than about 0.1 M, greater than about 0.2 M, greater thanabout 0.2 M, greater than about 0.3 M, greater than about 0.4 M, greaterthan about 0.5 M, greater than about 1.0 M, greater than about 1.1 M,greater than about 1.2 M, greater than about 1.3 M, greater than about1.4 M, greater than about 1.5 M, greater than about 1.6 M, greater thanabout 1.7 M, greater than about 1.8 M, greater than about 1.9 M, greaterthan about 2.0 M, greater than about 2.5 M, greater than about 3.0 M,greater than about 3.5 M, greater than about 4.0 M, or greater thanabout 4.5 M

Capturing Biomarkers.

A microneedle that is covalently or non-covalently attached to a probecan be inserted into a biological sample in situ, such as human skin,eye, intraoperative tissue, dermal capillaries, etc. A microneedlecovalently attached to a probe can also be inserted into a biologicalsample ex vivo, such as tissue extracted during a biopsy. The probe canbe allowed to hybridize or bind to a biomarker under physiologicalconditions of the biological sample for a specified period of time. Atemperature range of from about 20 to about 40 degrees Celsius,atmospheric pressure of 1, pH of 6-8, glucose concentration of 1-20 mM,atmospheric oxygen concentration, and earth gravity can be examples ofphysiological conditions for most subjects. The probe can be allowed tohybridize or bind to the biological sample for at least 1 minute, atleast 2 minutes, at least 3 minutes, at least 5 minutes, at least 10minutes, at least 15 minutes, at least 20 minutes, at least 25 minutes,at least 30 minutes, at least 45 minutes, at least 1 hour, at least 2hours, at least 3 hours, at least 5 hours, at least 10 hours, or atleast 24 hours. In some embodiments, the probe can be allowed tohybridize to the biological sample for no more than 1 minute, no morethan 2 minutes, no more than 3 minutes, no more than 5 minutes, no morethan 10 minutes, no more than 15 minutes, no more than 20 minutes, nomore than 25 minutes, no more than 30 minutes, no more than 1 hour, nomore than 2 hours, no more than 3 hours, nor more than 5 hours, or nomore than 10 hours. A microneedle with a covalently or non-covalentlylinked probe can be removed from the biological sample, for example, thehuman skin. A biomarker that is hybrized or bound to a probe can beisolated from a biological sample by removing the microneedle from thehuman skin.

Detecting Biomarkers.

Some embodiments of the invention are directed to detectingpolynucleotide biomarkers (e.g., mRNAs, DNA). In these embodiments, theprobes (e.g., oligonucleotide probes, polynucleotide probes) for one ormore specific biomarkers can be readily synthesized based on thesequences of the target biomarkers. Once nucleic acid biomarkers arebound to the probes on the microneedle device of the invention, they aretypically subjected to an amplification reaction (e.g., PCR, reversetranscription PCR) or are detected by a labeled tag. The labeled tag maybe directly linked to the probe that is attached to the microneedle or,in some cases, the labeled tag binds to the biomarker after it hasalready been captured by the probe attached to the microneedle.

Many methods routinely practiced in the art can be readily employed toamplify nucleic acid biomarkers obtained from a subject. These include,e.g., polymerase chain reaction (PCR) or reverse transcription PCR. Seegenerally PCR Technology: Principles and Applications for DNAAmplification (ed. H. A. Erlich, Freeman Press, NY, NY, 1992); PCRProtocols: A Guide to Methods and Applications (eds. Innis, et al.,Academic Press, San Diego, Calif., 1990); Mattila et al., Nucleic AcidsRes. 19, 4967 (1991); Eckert et al., PCR Methods and Applications 1, 17(1991); PCR (eds. McPherson et al., IRL Press, Oxford); and U.S. Pat.No. 4,683,202 (each of which is incorporated by reference for allpurposes). Other suitable amplification methods include the ligase chainreaction (LCR) (see Wu and Wallace, Genomics 4, 560 (1989), Landegren etal., Science 241, 1077 (1988), transcription amplification (Kwoh et al.,Proc. Natl. Acad. Sci. USA 86, 1173 (1989)), and self-sustained sequencereplication (Guatelli et al., Proc. Nat. Acad. Sci. USA, 87, 1874(1990)) and nucleic acid based sequence amplification (NASBA). Thelatter two amplification methods involve isothermal reactions based onisothermal transcription, which produce both single stranded RNA (ssRNA)and double stranded DNA (dsDNA) as the amplification products in a ratioof about 30 or 100 to 1, respectively. Once amplified, identity of thecaptured biomarkers can be then readily confirmed by standardtechniques, e.g., sequencing analysis, electrophoresis, etc.

Some embodiments of the invention use PCR to detect a biomarker that ishybridized, or otherwise connected to a probe. The amplification of abiomarker by PCR can be across several orders of magnitude, sometimesstarting from a single or a few copies of the target and generatingthousands to millions of copies of a particular DNA sequence. PCR canuse thermal cycling, comprising cycles of repeated heating and coolingof the reaction for DNA melting and enzymatic replication of the DNA.These thermal cycling operations can physically separate the two strandsin a DNA double helix at a high temperature in a process called DNAmelting. At a lower temperature, each strand can then be used as thetemplate in DNA synthesis by the DNA polymerase to selectively amplifythe target DNA. The selectivity of PCR can result from the use ofprimers (short DNA fragments) that are complementary to the DNA regiontargeted for amplification under specific thermal cycling conditions.

Primers containing sequences complementary to a biomarker of interestalong with a DNA polymerase can be used to achieve selective andrepeated amplification. As PCR progresses, the DNA generated can be usedas a template for replication, setting in motion a chain reaction inwhich the DNA template is exponentially amplified. PCR applications canemploy a heat-stable DNA polymerase, such as Taq polymerase, an enzymeoriginally isolated from the bacterium Thermus aquaticus. This DNApolymerase can enzymatically assemble a new DNA strand from thenucleotides, e.g., by using single-stranded DNA as a template and DNAoligonucleotides (also called DNA primers) for initiation of DNAsynthesis.

A PCR reaction can be performed directly on a microneedle that has beeninserted into a biological sample. For example the microneedle may beplaced in a tube or between two plates (e.g., glass slides) comprisingthe necessary reagents for a PCR reaction. A biomarker may be detachedfrom the needle and released into the PCR tube by a plurality ofdifferent methods. For example, a microneedle may be heated to releasethe biomarker from the microneedle, or a biomarker may spontaneously bereleased from the needle into the PCR solution. A PCR reaction can beperformed as described above and the PCR product can be analyzed usingstandard procedures, for example, electrophoresis, real-time PCR, andother procedures, such as described in PCR Technology: Principles andApplications for DNA Amplification (ed. H. A. Erlich, Freeman Press, NY,NY, 1992); PCR Protocols: A Guide to Methods and Applications (eds.Innis, et al., Academic Press, San Diego, Calif., 1990). Different PCRmethods can be used to analyze the sample, such as standard PCR methodsand Real-time PCR methods. Real-time PCR (RT-PCR) is a laboratorytechnique based on PCR, which can be used to amplify and simultaneouslyquantify a targeted DNA molecule. Real-time PCR can be combined withreverse transcription to quantify messenger RNA and non-coding RNA incells or tissues.

In some embodiments, a device of the disclosure can be furtherconfigured to comprise at least one compartment that can perform a PCRreaction. For example, a device of the disclosure may be configured tocomprise a plurality of microneedles or an array of microneedles and acompartment where a PCR reaction can be performed.

A PCR reaction can selectively amplify a biomarker that has beenhybridized to a particular microneedle or a PCR reaction can amplify aset of biomarkers that have been hybridized to a plurality ofmicroneedles. For example, 402 illustrates a surface of a device of theinvention comprising a plurality of microneedles that have beencontacted with a biological sample. Each “square” in 402 illustrates anindividual microneedle, wherein each individual microneedle comprises atleast one probe. Each probe in 402 can hybridize to a biomarker, or not.Each microneedle illustrated in 402 can be placed into a separate PCRtube and each PCR product can be analyzed individually. Alternatively, aplurality of microneedles illustrated in 402 can be placed into the samePCR tube for simultaneous analysis.

In some cases, a captured biomarker may be detected by using a labeledprobe capable of binding to the captured biomarker. In some cases, thelabeled probe binds to the biomarker after the biomarker has alreadybeen captured by a probe attached to a microneedle described herein. Insome cases, the microneedle is directly attached to a probe that istagged with a label designed to change its optical signal (eitherdecreasing or increasing in intensity) when bound to a biomarker.

The labeled probe may comprise a label (e.g., fluorophore, radioisotope,etc.) that can emit an optical signal. A fluorescent moiety can be afluorescent protein, such as a green fluorescent protein (GFP), readfluorescent protein (RFP), yellow fluorescent protein (YFP) orvariations thereof. In some cases, the labeled probe comprises a labelwith an optical signal that increases or decreases when the probe isbound to its target. A fluorescent moiety can be an RNA aptamer thatbinds fluorophores. An RNA-fluorophore complex can emit an opticalsignal that spans the visible spectrum, see Paige et al, Science 333,6042 (2011). For example, the “Spinach aptamer sequence” is an RNA mimicof GFP that can be configured to emit a fluorescent optical signal whenhybridized to a biomarker.

In some embodiments, a microneedle comprising a set of probes that arenot-covalently or covalently bound therein can be used to detect abiomarker in a subject. For example, a device of the disclosure can becontacted to the skin of a subject. The device can comprise apolynucleotide probe comprising an RNA-fluorophore moiety. TheRNA-fluorophore moiety can be configured to emit an optical signal, suchas a fluorescence signal, when hybridized to a biomarker of interest.

Protein or peptide biomarkers can be detected and quantified by any of anumber of methods well known to those of skill in the art forpolypeptide detection. These include assay formats such as protein PCRand ELISAs. Both local and systemic protein and peptide biomarkers canbe assayed using the microneedle array devices of the invention. Othermethods suitable for this purpose include analytic biochemical methodssuch as electrophoresis, capillary electrophoresis, high performanceliquid chromatography (HPLC), thin layer chromatography (TLC),hyperdiffusion chromatography, mass spectroscopy and the like, orvarious other immunological methods such as fluid or gel precipitinreactions, immunodiffusion (single or double), immunohistochemistry,affinity chromatography, immunoelectrophoresis, radioimmunoassay (RIA),immunofluorescent assays, Western blotting, dipstick, and the like. Fora general review of immunoassays, see also Methods in Cell BiologyVolume 37: Antibodies in Cell Biology, Asai, ed. Academic Press, Inc.New York (1993); Basic and Clinical Immunology 7th Edition, Stites &Terr, eds. (1991); IMMUNOASSAYS FOR THE 80s. Voller, A. et al (editors),Baltimore: University Park Press (1981); Maggio, et al,ENZYME-IMMUNOASSAY, Boca Raton: CRC Press pp 172-176 (1980) and Tijssen,Laboratory Techniques in Biochemistry and Molecular Biology: Practiceand Theory of Immunoassays, vol 15, Elsevier 1985. Reagents forperforming these assays with respect to any specific protein or peptidebiomarker (e.g., antibodies) can be readily obtained from commercialsources or generated by standard and routinely practiced techniques(e.g., hybridoma technology for producing monoclonal antibodies).

Binding interactions between a probe and a biomarker can be detectedusing a secondary detection reagent, such as a secondary antibody. Forexample, a “sandwich ELISA” can be used to detect binding of a biomarkerto an antibody probe. The binding of a biomarker to a probe can bedetected using a detection antibody that is specific to a differentepitope of the biomarker. The antibody used in the detection can be of adifferent isotype than the antibody used as a probe, for example, anantibody probe can be an IgG antibody (including any of the subtypes,such as IgG1, IgG2, IgG3 and IgG4), and a secondary antibody can be ofthe IgA, IgM). The detection antibody can be, for example conjugated toa detectable label, such as a fluorescent moiety or a radioactive label.An antibody based method of detection, such as an enzyme-linkedimmunosorbent assay (ELISA) method can be used to detect a binding of aprobe to a biomarker.

Protein and peptide biomarkers can also be assayed using protein PCRtechniques. For example, one assay suitable for detecting the biomarkersin these applications is the PCR-ELISA protocol. The assay employs astandard immunoassay procedure. A capture antibody is attached to thesurface of the microneedles of the devices. Instead of using a reporterenzyme for development of analytical signal, the secondary antibody isfused to a single stranded oligonucleotide that can then be amplifiedusing PCR. After insertion and capture of the biomarkers, the presenceof the biomarkers can be detected by addition of theoligonucleotide-labeled secondary antibody and subsequent PCR analysisof the conjugated tag.

Diagnostic Applications and Related Kits.

The devices and methods described herein are useful for detecting andcapturing biomarkers in various diagnostic applications. These include,e.g., diagnosis of skin diseases, detection of circulating geneticmarkers, and detection of protein or peptide biomarkers. As an example,the devices can be readily employed in the diagnosis of cutaneousmalignant melanoma (CMM) in subjects suspected to have or to be at riskof developing CMM. In these applications, probes for CMM-specificbiomarkers can be coupled to microneedles. Devices containing one ormore of the probe-conjugated microneedles can then be applied directlyto all moles (nevi) on a subject's skin. The devices are removed fromeach nevus, and any biomarkers captured by the microneedles are thenassayed either in situ on the device or after separation from thedevices. A device of the disclosure can be used clinically fordiagnostic and prognostic applications.

As another specific application, the devices and methods describedherein are employed in margin detection during skin cancer resectionsurgery. In these embodiments, the devices contain probe-conjugatedmicroneedles of different lengths. Detection and assaying of tumorspecific biomarkers with these devices can inform the surgeon about theextent of tumor invasion in the cutaneous, subcutaneous and underlyingspace. These applications obviate the iterative histopathologicalexamination that is currently required to determine margins during skintumor resection. In some cases, the method does not comprise biopsy ofthe skin.

A method and a device of the invention can be used in the diagnosisand/or treatment of a skin conditions. These methods can comprisecontacting the microneedle with the skin tissue of a subject. A skincondition can be a benign condition, a pre-malignant condition, or amalignant condition. A skin condition can be a healthy condition.Non-limiting examples of skin conditions include: skin cancer, such asmelanoma and Mohs skin cancer; onchocerciasis; lupus; rubeola (measles);hemangioma; psoriasis; rosacea; seborrheic eczema; hives, vitiligo;warts; necrotizing fasciitis; cutaneous candidiasis; carbuncle;cellulitis; hypohidrosis; impetigo; cutis laxa; decubitus ulcer;erysipelas; dyshidrotic eczema; canker sore; mole(s); herpes stomatitis;ichthyosis vulgaris; acne; cold sore; dermatomyositis; molluscumcontagiosum; acrodermatitis; sebaceous cyst; seborrheic keratosis;pilonidal sinus; keloid; lichen planus; actinic keratosis; stasisdermatitis; skin corns and calluses; eczema; tinea versicolor;pemphigoid; ulcers; or shingles. A method and a device of the inventioncan be used in the diagnosis and treatment of a plurality of ocularconditions such as uveitis, dry eye disease, retinal disease, glaucoma,inflammatory diseases. A device of the invention can be used to stage acancer. A cancer can be staged as stage 0, stage I, stage II, stage III,or stage IV.

A method and a device of the invention can also be used in the diagnosisand/or treatment of an eye conditions, such as, for example, cornealsurface inflammation, uveitis, or dry eye disease. These methods cancomprise contacting the microneedle with the eye tissue of a subject,for example, by contacting the sub-conjunctival space. An eye conditioncan be a benign condition, a pre-malignant condition, or a malignantcondition. A eye condition can be a healthy condition. Non-limitingexamples of eye conditions include: retinoblastoma; cutaneous orintraocular (eye) melanoma; retinitis pigmentosa (RP); diabeticretinopathy; glaucoma, (including open-angle glaucoma (e. g., primaryopen-angle glaucoma), angle-closure glaucoma, and secondary glaucomas(e. g., pigmentary glaucoma, pseudoexfoliative glaucoma, and glaucomasresulting from trauma and inflammatory diseases)), retinal detachment,age-related or other maculopathies, age-related macular degeneration,photic retinopathies, surgery-induced retinopathies, toxicretinopathies, retinopathy of prematurity, retinopathies due to traumaor penetrating lesions of the eye, inherited retinal degenerations,surgery-induced retinopathies, toxic retinopathies, retinopathies due totrauma or penetrating lesions of the eye. Specific exemplary inheritedconditions of interest include, but are not necessarily limited to,Bardet-Biedl syndrome; Congenital amaurosis; Cone or cone-rod dystrophy;Congenital stationary night blindness; Macular degeneration; Opticatrophy; Syndromic or systemic retinopathy; and Usher syndrome.

A method and a device of the invention may be used to monitor theexpression of a biomarker in a surgical procedure or in a cosmeticprocedure. The device and methods of the disclosure can be used to, forexample perform a biopsy of delicate tissues, such as the eye tissue orbrain tissue. In some cases, the microneedle device can be contactedwith the tissue or biological sample of the subject during anintraoperative procedure. The tissue or biological sample can beobtained from an organ selected from the group consisting of brain,heart, breast, liver, pancreas, spleen, bladder, stomach, lung, uterus,cervix, prostate, kidney, intestine, appendix, and colon. In furthercases, the microneedles can be contacted to the margins of a tumorbefore or after the tumor is removed from the subject.

As a further example of the diagnostic applications of the invention,the methods and devices described herein can be used in the detection ofsystemic and circulating genetic biomarkers in a bodily fluid (e.g., theblood stream) of a subject. Many diseases (e.g., Down's syndrome) areknown to have genetic (e.g., mRNA) biomarkers that circulate in theblood. By extending the lengths of the microneedles in the devices ofthe invention, probes for the biomarkers are capable of specificallybinding to such biomarkers by penetrating into and accessing dermal orsubcutaneous capillaries. Any other nucleic acid biomarkers that areknown to be present in the blood can also be detected in a similarmanner. For example, the microneedles on a device of the disclosure canpenetrate the skin of a subject in situ, and be in contact with thedermal capillaries of the subject. Dermal capillaries can be thesmallest blood vessels in the body of a subject and the endotheliallinings of dermal capillaries can be about one cell layer thick. Adevice of the disclosure can penetrate the membrane of one, or aplurality of dermal capillaries when the device is contacted to a skinof a subject. In some embodiments, a device of the disclosure may beutilized to test biomarkers circulating in the bloodstream without theremoval of a blood sample from a subject. In some cases, a device of thedisclosure can detect fetal or maternal biomarkers of a condition,including pregnancy associated biomarkers. In some cases, a device ofthe invention can detect biomarkers circulating in the bloodstream suchas proteins, hormones, vitamins, co-factors, or polynucleotides.

Non-limiting examples of genetic conditions that can be diagnosed with amethod and a device of the invention based on a polynucleotide biomarkerinclude: cystic fibrosis; Duchenne muscular dystrophy; Haemochromatosis;Tay-Sachs disease; Prader-Willi syndrome; Angelman syndrome;neurofibromatosis; phenylketonuria; Canavan disease; Coeliac disease;Acid beta-glucosidase deficiency; Gaucher; Charcot-Marie-Tooth disease;color blindness; Cri du chat; polycystic kidney disease; acrocephaly;familial adenomatous polyposis; adrenal gland disorders; amyotrophiclateral sclerosis (ALS); Alzheimer's disease; Parkinson's disease;anemia; ataxia; ataxia telangiectasia; autism; bone marrow diseases;Bonnevie-Ullrich syndrome; brain diseases; von Hippel-Lindau disease;congenital heart disease; Crohn's disease; dementia; myotonic dystrophy;Fabry disease; fragile X syndrome; galactosemia; genetic emphysema;retinoblastoma; Pendred syndrome; Usher syndrome; Wilson disease;neuropathies; Huntington's disease; immune system disorders; gout;X-linked spinal-bulbar muscle atrophy; learning disabilities;Li-Fraumeni syndrome; lipase D deficiency; Lou Gehrig disease; Marfansyndrome; metabolic disorders; Niemann-Pick; Noonan syndrome;Osteopsathyrosis; Peutz-Jeghers syndrome; Pfeiffer syndrome; porphyria;progeria; Rett syndrome; tuberous sclerosis; speech and communicationdisorders; spinal muscular atrophy; Treacher Collins syndrome;trisomies; and monosomies.

In some cases, the probes of the invention can be used to detect acondition of the immune system. A device of the invention can be used inallergy testing to confirm or to rule out allergies. In some cases adevice of the invention can be used to analyze an allergy panel.Non-limiting examples of immune disorders include: HIV, diabetes,Parkinson's disease, Alzheimer's, rheumatoid arthritis, lupus, cancer,multiple sclerosis, inflammatory bowel disease, psoriasis, scleroderma,autoimmune thyroid disease, vasculitis, pernicious anemia, severecombined immunodeficiency (SCID), DiGeorge syndrome, hyperimmunoglobulinE syndrome, common variable immunodeficiency, chronic granulomatousdisease, Wiskott-Aldrich syndrome, autoimmune lymphoproliferativesyndrome (ALPS), hyper IgM syndrome, leukocyte adhesion deficiency(LAD), NF-κB essential modifier (NEMO) disorders, selectiveimmunoglobulin A deficiency, X-linked agammaglobulinemia, X-linkedlymphoproliferative disease, ataxia-telangiectasia, seasonal allergy,mastocytosis, perennial allergy, anaphylaxis, food allergy, allergicrhinitis, and atopic dermatitis.

In some embodiments, the devices and methods of the invention can beused to diagnose a variety of biomarkers associated with a plurality ofcancers. Non-limiting examples of cancers can include: acutelymphoblastic leukemia, acute myeloid leukemia, adrenocorticalcarcinoma, AIDS-related cancers, AIDS-related lymphoma, anal cancer,appendix cancer, astrocytomas, basal cell carcinoma, bile duct cancer,bladder cancer, bone cancers, brain tumors, such as cerebellarastrocytoma, cerebral astrocytoma/malignant glioma, ependymoma,medulloblastoma, supratentorial primitive neuroectodermal tumors, visualpathway and hypothalamic glioma, breast cancer, bronchial adenomas,Burkitt lymphoma, carcinoma of unknown primary origin, central nervoussystem lymphoma, cerebellar astrocytoma, cervical cancer, childhoodcancers, chronic lymphocytic leukemia, chronic myelogenous leukemia,chronic myeloproliferative disorders, colon cancer, cutaneous T-celllymphoma, desmoplastic small round cell tumor, endometrial cancer,ependymoma, esophageal cancer, Ewing's sarcoma, germ cell tumors,gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor,gastrointestinal stromal tumor, gliomas, hairy cell leukemia, head andneck cancer, heart cancer, hepatocellular (liver) cancer, Hodgkinlymphoma, Hypopharyngeal cancer, intraocular melanoma, islet cellcarcinoma, Kaposi sarcoma, kidney cancer, laryngeal cancer, lip and oralcavity cancer, liposarcoma, liver cancer, lung cancers, such asnon-small cell and small cell lung cancer, lymphomas, leukemias,macroglobulinemia, malignant fibrous histiocytoma of bone/osteosarcoma,medulloblastoma, melanomas, mesothelioma, metastatic squamous neckcancer with occult primary, mouth cancer, multiple endocrine neoplasiasyndrome, myelodysplastic syndromes, myeloid leukemia, nasal cavity andparanasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma,non-Hodgkin lymphoma, non-small cell lung cancer, oral cancer,oropharyngeal cancer, osteosarcoma/malignant fibrous histiocytoma ofbone, ovarian cancer, ovarian epithelial cancer, ovarian germ celltumor, pancreatic cancer, pancreatic cancer islet cell, paranasal sinusand nasal cavity cancer, parathyroid cancer, penile cancer, pharyngealcancer, pheochromocytoma, pineal astrocytoma, pineal germinoma,pituitary adenoma, pleuropulmonary blastoma, plasma cell neoplasia,primary central nervous system lymphoma, prostate cancer, rectal cancer,renal cell carcinoma, renal pelvis and ureter transitional cell cancer,retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcomas, skincancers, skin carcinoma merkel cell, small intestine cancer, soft tissuesarcoma, squamous cell carcinoma, stomach cancer, T-cell lymphoma,throat cancer, thymoma, thymic carcinoma, thyroid cancer, trophoblastictumor (gestational), cancers of unkown primary site, urethral cancer,uterine sarcoma, vaginal cancer, vulvar cancer, Waldenströmmacroglobulinemia, and Wilms tumor.

A device of the disclosure can be used for an independent diagnosticmethod, or as a companion diagnostic to inform a therapeutic treatment.The microneedle and methods of the disclosure provide a method ofdiagnosis and a method of treatment that is minimally-invasive, fast,and accurate. In addition, the devices and methods disclosed herein canprovide a portable, pain-free, and affordable diagnosis to a pluralityof subjects. A subject of the invention can be of any age, including,for example, elderly adults, adults, adolescents, pre-adolescents,children, toddlers, and infants. A subject of the invention can be amammal, a bird, a fish, a reptile, or an amphibian. Non-limitingexamples of a subject include humans, primates, dogs, cats, horses,pigs, and mice.

A subject can provide a plurality of biological samples for an analysiswith a microneedle of the invention. An analysis of a biological sampleof a subject can be performed in situ or ex vivo. For example, an insitu analysis can comprise contacting a microneedle of the inventiondirectly to the skin of a subject. An ex vivo analysis can comprisecontacting a microneedle of the invention to a biopsy tissue. In someembodiments, about 1 mg, about 5 mgs, about 10 mgs, about 15 mgs, about20 mgs, about 25 mgs, about 30 mgs, about 35 mgs, about 40 mgs, about 45mgs, about 50 mgs, about 55 mgs, about 60 mgs, about 65 mgs, about 7mgs, about 75 mgs, about 80 mgs, about 85 mgs, about 90 mgs, about 95mgs, or about 100 mgs of a biological sample are required for abiomarker analysis with a microneedle and method of the invention.

In some embodiments, no more than about 1 mg to about 5 mgs, no morethan about 1 mg to about 10 mgs, no more than about 1 mg to about 20mgs, no more than about 1 mg to about 30 mgs, no more than about 1 mg toabout 40 mgs, no more than about 1 mg to about 50 mgs, no more thanabout 50 mgs to about 60 mgs, no more than about 50 mgs to about 70 mgs,no more than about 50 mgs to about 80 mgs, no more than about 50 mgs toabout 90 mgs, no more than about 50 mgs to about 100 mgs, no more thanabout 100 mgs to about 1 gram, no more than about 100 mgs to about 2grams, no more than about 100 mgs to about 3 grams, no more than about100 mgs to about 4 grams, no more than about 100 mgs to about 5 grams,no more than about 100 mgs to about 6 grams, no more than about 100 mgsto about 7 grams, no more than about 100 mgs to about 8 grams, no morethan about 100 mgs to about 9 grams, no more than about 100 mgs to about10 grams, no more than about 1 gram to about 2 grams, no more than about1 gram to about 3 grams, no more than about 1 gram to about 4 grams, nomore than about 1 gram to about 5 grams, no more than about 1 gram toabout 6 grams, no more than about 1 gram to about 7 grams, no more thanabout 1 gram to about 8 grams, no more than about 1 gram to about 9grams, or no more than about 1 gram to about 10 grams of biologicalsample are required by the methods and microneedle of the invention.

In some embodiments, additional assays are used to validate a diagnosisthat is made based on the identification of biomarker identified with amicroneedle and a method of the disclosure. Non-limiting examples ofassays that can validate a biomarker include: a) assays that evaluatethe interactions of proteins with DNA, such as DNase footprinting assayand gel shift assays; b) assays that evaluate the integrity of an RNAmolecule, such as nuclear run-on assays; c) end point assays, which canmeasure quantitatively or qualitatively the end result of an assay; d)kinetic assays, which can evaluate readings of data points at multipletime intervals and compare the kinetics of a biological process; e)semi-quantitative assays, which provide a read-out that can bequantitated within a context, such as western-blots, clotting andagglutination assays; f) immunoassays, which evaluate the response of anantigen antibody binding type of reaction; g) enzyme activity assays,which test function and activity; h) colony forming assays, which cantest the ability of a cell to proliferate and differentiate; i) countingassays, such as flow cytometry assays; and j) a plurality of PCR assays,such as real-time PCR.

Further, the methods of the disclosure can further comprise detectingone or more biomarkers from a reference tissue obtained from thesubject. For example, the method may comprise detecting biomarkers froma sample tissue and a reference tissue. The reference tissue can be abenign tissue. In some cases, the reference tissue is tissue from thesame organ or region as the sample tissue. In some cases, the sampletissue comprises tissue suspected to have a disease or disorder (e.g.,malignancy) and the reference tissue comprises tissue of the same organthat is known to not have the disease or disorder. In some cases, thedetected levels of the biomarker in the sample tissue can be furthercompared to the detected levels of the biomarker in the referencetissue.

In some cases, a device of the disclosure can be used during surgery toremove tumors and/or to identify tumor margins. A device of theinvention can be used to characterize tissues and tissue margins withdistinct morphologies in situ or ex vivo. For example, in Mohs skincancer surgery, where tissue is usually removed, sectioned and evaluatedby histological methods a device of the invention could be used toanalyze the margins of the tumor. In some cases, a device of theinvention can be used to identify the metastasis of a cancer byanalyzing the margins of a tissue.

A device and methods of the disclosure can be utilized in personalizedmedicine applications. A subject can provide a quantity of, for example,a skin sample to a clinician. The clinician may use a device of thedisclosure to test for a plurality of biomarkers associated with theskin of the subject. The clinician may use the identified biomarkers todetermine the expected efficacy of a treatment on a particular subject.A device and a method of the disclosure may also be used to monitor theresponse of a subject to a particular treatment, for example, bymonitoring an increase or a decrease in the expression of a biomarker. Aclinician may rely on an increase or a decrease of a biomarkerexpression level to determine an effectiveness of a treatment. A deviceof the invention can be used to quantitatively measure the expression ofa biomarker. For example, quantitative PCR can be used to amplify thenumber of copies of a biomarker that has hybridized to a polynucleotideprobe on a microneedle. A microneedle that has not been contacted orhybridized with a biological sample can be used a negative control. Amicroneedle with a known amount of a standard control, such as ahousekeeping gene, can be used as a positive control for the reaction.Quantitative PCR can be performed as described in The PCR Technique:Quantitative PCR, James W. Larrick, June 1997, Eaton Publishing.

A device and methods of the disclosure can be used in biodefense.Biodefense can comprise detecting the release or dissemination ofbiological or chemical agents. These agents can be bacteria, viruses, ortoxins, and may be in a naturally occurring or a human-modified form. Adevice of the disclosure can be used to detect a plurality of biomarkersassociated with pathological agents that can be used in a biologicalwar.

An agent of biological and/or chemical warfare can include anybiological and/or chemical entity that can be used as a weapon, e.g., tocause terror, chaos, disease, discomfort and/or death. Non-limitingexamples of chemical and biological agents include anthrax; small pox;tularemia; avian flu; plague; HIV; ebola; foot and mouth disease; ricin;neurotoxic organophosphates; cyanide; vesicants (or blister agents) suchas mustard gas and Lewisite; choking agents, such as chlorine andphosgene; nerve agents, such as sarin, Tabun, Soman, and VX;hallucinogens, such as BZ; pesticides such as parathion, malathion, andazinphosmethyl; and derivatives or combinations thereof.

The invention further provides kits for carrying out the diagnosticapplications described herein. The kits typically contain a microneedledevice comprising one or more microneedles. In some kits, themicroneedles are already conjugated with probes specific for detectingone or more biomarkers. In some other kits, probes for detecting one ormore biomarkers and reagents for their conjugation to the microneedlesare provided as separate components. Some kits of the invention areintended for detecting and capturing one specific biomarker for a knowndisease or disorder (e.g., Plakophilin-3 mRNA for gastrointestinalcancer). In these kits, the microneedles of the device usually areconjugated or to be conjugated with the same probe molecule (e.g., anoligonucleotide complementary to the target biomarker). Some other kitsof the invention are designed for detection of a plurality of biomarkersimplicated in one or more diseases or disorders. In these kits,different probes for the different biomarkers are conjugated or providedfor conjugation to the microneedles of the device. In addition to themicroneedle device and probes, the kits of the invention can alsoinclude other reagents for applying the device to a subject and foranalyzing the captured biomarker (e.g., reagents for PCR amplificationof biomarkers). The kits can further contain instructions (e.g., on aninstruction sheet in the kits or packaging material of the kits) forusing the kits to carry out the intended diagnostic applications.

Devices of the invention can be packaged as a kit. In some embodiments,a kit includes written instructions on the use of the device fortreatment of a condition, such as basal cell carcinoma. The writtenmaterial can be, for example, a label. The written material can suggestconditions and methods of using the microneedle and additional reagentscomprised in the kit. The instructions provide the subject and thesupervising physician with the best guidance for achieving the optimaldetection of a biomarker.

A kit of the disclosure may comprise a device described herein and a setof reagents for a polymerase chain reaction. A kit of the disclosure maycomprise a device described herein and a set of reagents for an ELISAassay. A kit may be designed for the identification of a specificcondition, such as basal cell carcinoma or a kit may be designed for thesimultaneous diagnosis of a plurality of conditions, such as squamouscell carcinoma, Kaposi's sarcoma, melanoma, basal cell carcinoma, andactinic keratosis (a precursor to squamous cell carcinoma). In somecases, a kit of the disclosure may comprise a device of the inventionand written material. In some cases, a kit of the disclosure maycomprise a device of the invention, a set of probes, and a set ofreagents that can be used to link the probes to the microneedles.

A kit of the disclosure can comprise positive and negative controls. Apositive control can be, for example, a sample comprising a knownbiomarker. A positive control can be a polynucleotide of known sequence,such as a polynucleotide of a known SNP associated with basal cellcarcinoma. A negative control can be, for example, a scrambledpolynucleotide sequence. In some cases, the kit may comprise reagents(e.g., polypeptide, polynucleotide) of known concentrations orquantities that can be used to prepare a standard curve in order toquantify the amount of biomarker present in a tissue or biologicalsample. Such reagents may also be used in conjunction with any methodprovided herein.

Animal Models.

Many drugs, treatments, and cures for diseases can be developed with theuse of animal models. An animal model can be a live animal used duringthe research and investigation of disease. The pharmaceuticaldevelopment of a drug can include the investigation of toxic and adverseside effects that are not detected in cellular assays. An animal modelcan be used to appraise efficacy, absorption, metabolism, distribution,excretion, toxicity, pharmacology, and side effects of a pharmacologicaltreatment. A device and the methods of the invention can be used toidentify the response of a biomarker on an animal model to apharmacological treatment.

An animal model can provide guidance for the selection ofpharmacological compounds for further evaluation. An animal model can,for example, provide guidance for pharmacokinetic/metabolic studies inhumans. An animal model can be used, for example, for the evaluation ofabsorption, distribution, metabolism, excretion, and toxicology (ADMET)parameters supporting an Investigational New Drug (IND) application.Studies in an animal model can, for example, lead to the selection ofcompounds to be further evaluated in pre-clinical and clinical trials.

Clinical Intervention and Clinical Trials

A device of the disclosure can be used in a clinical trial. For example,a device of the disclosure may be used during a surgical procedure toidentify a biomarker in a tissue that is difficult to reach, or a tissuethat cannot be excised and removed for a biopsy. For example, a deviceof the disclosure can be applied to the detection of a biomarker in acardiovascular tissue, a brain tissue, or an internal organ that isexposed to a clinician during a surgical procedure. In some cases, adevice of the invention can prevent medical complications that otherwisecan arise when a clinician removes tissue from a subject for a biopsy. Adevice of the disclosure can be used, for example, in a routine medicalconsultation, during a surgical procedure, or by a subject at asubject's home. A device of the invention can be used, for example, incardiovascular surgery or eye surgery.

A device of the disclosure can be used in the design of a clinical trialprotocol. For example, a device of the disclosure can be used inconjunction with a clinical trial to detect the levels of a biomarkerand to monitor the response of a subject to a pharmacological treatment.A device of the disclosure can provide guidelines for the design,conduct, and analyses of pre-clinical development and clinical trials.The disclosure provides a method of identifying and quantifyingbiomarkers that can be selected for the optimal administration oftherapeutically effective compounds.

A device of the disclosure can be used in predicting the subject'sresponse to distinct drug dosages in a clinical trial. For example, bymonitoring increases or decreases in the levels of a biomarker, such asa biomarker of retinal disease, in an ocular tissue the methods anddevices of the invention can establish an efficacy of a pharmacologicaltreatment for the retinal disease. In some cases, a clinical trial for atherapeutic agent is conducted or altered based on the detection of abiomarker with a device of the disclosure. In some cases, the methodsand devices of the disclosure can be used to compare a treatment beingevaluated in a clinical trial to a known standard-of-care treatment.

Clinical trials typically proceed through several steps, includingpre-clinical studies, pilot studies, safety screening studies, andefficacy evaluation studies. For a drug to be approved and marketed, allmilestones specified in a clinical trial protocol must often be met,including, demonstration of efficacy within a proposed confidenceinterval, and inclusion of a significant number of individuals todemonstrate the statistical power of the invention. Non-limitingexamples of applications of the invention include the monitoring of aplurality of biomarkers throughout the course of a clinical trial. Adevice and methods of the disclosure can also be used to monitor howdistinct pharmacological treatments can affect the expression ofbiomarkers in the early stages of a clinical trial (pre-clinical andphase I).

A device and a method of the disclosure can also be used to predictchanges in underlying cellular pathways affected by a pharmacologicaltreatment based on the expression levels of the identified biomarkers.Non-limiting examples of pharmacodynamic and pharmacokinetic parametersof a pharmacological treatment that can affect an underlying cellularpathway include: a) the amount of drug administered, which can berepresented as a dose D; b) the dosing interval, which can berepresented as τ; c) the apparent volume in which a drug is distributed,which can be represented as a volume of distribution V_(d), whereV_(d)=D/C₀; d) the amount of drug in a given volume of plasma, which canbe represented as concentration C₀ or C_(ss), where C₀ or C_(ss)=D/Vd;e) the half-life of a drug t_(1/2), where t_(1/2)=ln(2)/k_(e); f) therate at which a drug is removed from the body k_(e), wherek_(e)=ln(2)/t_(1/2)=CL/V_(d); g) the rate of infusion required tobalance the equation K_(in), where K_(in)=C_(ss).CL; h) the integral ofthe concentration-time curve after administration of a single dose,which can be represented as AUC_(0-∞), wherein ∫₀ ^(∞) C dt, or insteady-state, which can be represented as AUCτ, _(ss), wherein ∫_(t)^(t+π) C dt; i) the volume of plasma cleared of the drug per unit time,which can be represented as CL (clearance), whereinCL=V_(d).k_(e)=D/AUC; j) the systemically available fraction of a drug,which can be represented as f, where

${f = \frac{{AUC}_{{po} \cdot {Div}}}{{AUC}_{{iv} \cdot {Dpo}}}};$

k) the peak plasma concentration of a drug after administrationC_(max); 1) the time taken by a drug to reach C_(max), t_(max); m) thelowest concentration that a drug reaches before the next dose isadministered C_(min); and n) the peak trough fluctuation within onedosing interval at steady state, which can be represented as

%  PTF = 100.${\frac{\left( {C_{\max,{ss}} - C_{\min,{ss}}} \right)}{C_{{av},{ss}}}\mspace{14mu}{where}\mspace{14mu} C_{{av},{ss}}} = {\frac{{AUC}_{\tau,{ss}}}{\tau}.}$

EXAMPLES

The following examples are provided to further illustrate the inventionbut not to limit its scope.

Example 1. Detecting ssDNA in Solution with Polycarbonate ImmobilizedProbes

This Example describes polycarbonate functionalized by a DNA probe andits utilizations for specific capture of ssDNA from solution.

Attachment of DNA probes to polycarbonate: The surface of polycarbonatewas first nitrated and reduced to introduce amino groups which were usedfurther to attach commercially available thiol/amino bifunctional linker(FIG. 1). DNA with 3′ thiol modification was subsequently coupled to thelinker tethered to the polycarbonate.

Visualization of 5′-Cy5 modified DNA attached to polycarbonate: Tovisualize the DNA on the surface of the polycarbonate, DNA modified withone Cy5 at the 5′ and thiol at the 3′ was attached using chemistrydescribed above. Single layer 5′Cy5-DNA was imaged with confocalmicroscopy (FIG. 2) showing successful functionalization of the surface.In vitro capture of ssDNA on polycarbonate modified with specific DNAprobe: In order to test the ability of DNA probe attached to thepolycarbonate to specifically capture (hybridize) ssDNA from thesolution, polycarbonate disks were modified with DNA probe complementaryto region of circular single stranded DNA (ssDNA, about 3.4 kb in size).Upon incubation of the modified disks with ssDNA solution, disks werethoroughly washed, hybridized ssDNA was released from the disks bydenaturation at elevated temperature and quantified by quantitative PCR(qPCR). Amount of the DNA captured on the polycarbonate modified withspecific DNA probe was expressed as ratio to the capture onpolycarbonate modified with linker only—background control (FIG. 1).

Results obtained from the study are summarized in TABLE 1. The data showthat the ssDNA target was successfully enriched on specific probe at allssDNA concentrations tested (concentrations varying over 6 logs).

TABLE 1. Capture efficiency as ratio of DNA recovery from disks withspecific probe to recovery from disks modified with linker only.

TABLE 1 Experiment/negative ssDNA sample conc. control DNA recovery 1.66× 10⁻¹⁸ M (1.66 aM) >4 1.66 × 10⁻¹⁶ M (166 aM) >3 1.66 × 10⁻¹⁴ M (16.6fM) 11.4 1.66 × 10⁻¹² M (1.66 pM) 5

Materials and methods used in the study are described in detail below.

Polycarbonate derivatization: polycarbonate was shaken in aqueous 30%HNO3 solution for 30 min at 65° C. and then washed extensively withwater. Nitrated polycarbonate (step 1) was immersed into 10% solution ofNaBH4 in water and shaken at room temperature overnight and finallywashed extensively with water. Amino-modified polycarbonate (step 2) wasimmersed in 6.4 mM solution of Sulfo-GMBS (Pierce) in PBS pH 7.2 andshaken for 1.5 h at room temperature and finally washed extensively withwater.

DNA thiol group deprotection: DNA was purchased from IDT with ThiolModifier C3 S—S at the 3′ end. To deprotect the thiol modification, 5 μLof 100 μM DNA solution in water was treated with 25 μL of 100 mM2-mercaptoethanol in PBS pH 8.1 for 30 min at room temperature. Topurify the deprotected DNA, 600 μL of PN buffer from Qiagen nucleotideremoval kit was added to the reaction mixture followed by addition ofisopropyl alcohol (250 μL). The solution was applied onto Qiagennucleotide removal kit silica-column and washed according to kit'sinstructions followed by elution with 35 μL of PBS pH 7.2. DNAattachment to the polycarbonate: Solution of deprotected thiol modifiedDNA (step 4) was immediately applied onto polycarbonate modified withmaleimide linker (step 3) and incubated at 37° C. in a humidifiedatmosphere for 45 min then washed extensively with water and dried inair.

Visualization of Cy5 modified DNA: 25 mer DNA modified with one Cy5 atthe 5′ end and thiol modification at the 3′ end was attached to thesurface of the polycarbonate using chemistry described above. Afterextensive washing with water and drying, polycarbonate was mounted ontomicroscope slide and imaged with confocal microscope using Far Redfilter. The edge of DNA modified area on the surface was localized andimaged, clearly showing effect of modification with Cy5 labeled DNAmonolayer.

Capture of ssDNA on polycarbonate functionalized with specific DNAprobe: DNA probe with thiol modification at 3′(5′-CAAGTTTGCCTTTAGCGTCAGACTGTATTTTTTTT/ThioMC3/-3′) (SEQ ID NO:1) wasattached to polycarbonate disks using procedure described above (steps1-4). Disks for negative control experiments were modified with linkeronly (steps 1-3). Disks were immersed in solution of circular ssDNAisolated from filamentous phage (for DNA concentrations see TABLE 1) inSSC buffer 3× (150 mM NaCl, 15 mM Sodium Citrate) and incubated for 10min at 37° C. Disks were washed subsequently with three washing buffers.Washing buffer 1: 1×SSC+0.03% SDS; washing buffer 2: 0.2×SSC, washingbuffer 3: 0.05×SSC. After washing, disk were immersed in minimal volumeof sterile distilled water and heated at 90° C. for 2 minutes afterwhich still warm water was removed from disks. ssDNA in aliquot of watersolution was quantified using qPCR with primers specific for p3 gene offilamentous phage.

Example 2. Conjugation of DNA Probes to Stainless Steel Surfaces

This Example describes attachment of DNA oligomer probes containing a 3′thiol modification to stainless steel surfaces coated in gold.Attachment of DNA probe to gold-coated stainless steel: Gold surfaceswas readily modified by attaching thiol-derivatized single-stranded DNA.The sulfur atom of the thiolated DNA forms a covalent bond with gold.Visualization of DNA to gold-coated stainless steel: To visualize theDNA on the gold-coated surface of the stainless steel sample, a fill-inPCR reaction was performed to synthesize the complementary strand of thesingle-stranded DNA using fluorescently labeled dUTP. The resultingdouble-stranded oligomers contained multiple copies of Chromatide® AlexaFluor® 488-5-dUTP, which fluoresces in the green channel, similar tofluorescein. Fluorescence microscopy demonstrates successfulfunctionalization of the gold-coated stainless steel surface (FIG. 3).

Materials and methods used in the study are described in detail below.

DNA thiol group deprotection and conjugation to stainless steel: DNA waspurchased from IDT with Thiol Modifier C3 S—S at the 3′ end. Todeprotect the thiol modification, 5 μL of 100 μM DNA solution in waterwas treated with 25 μL of 100 mM 2-mercaptoethanol in PBS pH 8.1 for 30min at room temperature. To purify the deprotected DNA, 600 μL of PNbuffer from Qiagen nucleotide removal kit was added to the reactionmixture followed by addition of isopropyl alcohol (300 μL). The solutionwas applied onto Qiagen nucleotide removal kit silica-column and washedaccording to kit's instructions followed by elution with 24 μL of TEbuffer (10 mM Tris; 1 mM EDTA), pH 7.2.

A solution of deprotected thiol-modified DNA was immediately appliedonto the gold surface of a piece of stainless steel (approximately 5mm×2.5 mm). The solution was incubated at 37° C. in a humidifiedatmosphere for 16-20 hours and then washed extensively with waterfollowed by air drying.

Fill-in reaction to amplify fluorescent signal: A 75 bp single-strandedDNA oligo with a thiol derivatization at the 3′ end(5′-GCATGCATGCATGCATGCATGCATGCATGCATGCATGCATGCGCCTGTGGGCGACTAAATTCCGTTAAAGCCGGC/ThiolMC3/-3′) (SEQ ID NO:2) was attached to thegold-coated surface of the sample of stainless steel. After washing anddrying, the stainless steel sample was placed in a 0.5 mL tube. 48.5 pLof dH2O and 1.5 pL of a 10 pM primer complimentary to the 3′ end of thethiol-derivatized DNA were then added. The mixture was incubated at 55°C. for 2 minutes to allow the primer to anneal to the single-strandedDNA. The mixture was then allowed to return to room temperature.

The fill in reaction was then performed using a Chromatide® Alexa Fluor®488-5-dUTP purchased from Invitrogen. The following components wereadded to the mixture in step 3: 2 pL of 10 mM dNTP mix (dATP, dGTP,dCTP), 7.5 pL of 10× Klenow fragment buffer (New England Biolabs), 9.5pL dH2O, 4 pL of 1 mM 488-5-dUTP and 2 pL of Klenow fragment (NewEngland Biolabs). The reaction was incubated for 30 minutes at 37° C.The stainless steel sample was removed from the tub, washed extensivelywith water and allowed to air dry.

Visualization of Chromatide® Alexa Fluor® modified DNA: Once the sampleof stainless steel had dried, it was mounted onto a microscope slide andimaged with a fluorescent microscope using the green filter. The edge ofthe DNA modified area on the stainless steel was localized and imaged,clearly demonstrating conjugation of the DNA to the gold-coated surface.

Example 3. Enhanced Binding of Oligonucleotides to the Surface of MetalMicroneedle Arrays

To further facilitate the attachment of DNA oligomer probes containing a3′ thiol modification to stainless steel surfaces coated in gold, a NaCltitration was introduced. The protocol in EXAMPLE 2 described above wasperformed with the addition of increasing concentrations of NaCl. Theamount of ssDNA coupled to the surface increased proportionally to theamount of NaCl added, and an approximately 10-fold increase was observedat the 1M NaCl concentration (FIG. 7). The quantitation of DNA bound togold surface was determined using a Quanti-iT Green ssDNA Reagent Kit(Invitrogen).

Example 4. Methods of Diagnosis

Missing a melanoma diagnosis is a significant concern fordermatologists. The consequences of a missed diagnosis can bedevastating for patients, costly for insurance companies, and damagingto physicians, therefore the early detection of melanoma is critical.Yet, the invasive nature of a standard biopsy procedure can deter aphysician from pursuing a biopsy on a tissue that appears to be benign.FIGS. 5 and 6 illustrate the non-invasive diagnosis of melanoma with thedevices and methods of the disclosure. 501-505 describes the surface of603 in greater detail.

FIG. 5 illustrates a process whereby a DNA probe designed to detect abiomarker of melanoma hybridizes to the desired biomarker. 501illustrates a single DNA probe attached to the gold surface of amicroneedle. The DNA probe in 501 comprises a single nucleotidepolymorphism that selectively hybridizes to an RNA associated withmelanoma. The DNA probe in 501 was covalently linked to the gold surfacein the microneedle as described in Example 2. 505 illustrates abright-field and a fluorescent field image of the surface of a device ofthe invention, comprising a plurality of microneedles with covalentlylinked DNA probes. When the device touches the skin of the subject, themicroneedles gently disrupt the membrane of the cells in the skin beingcontacted. This process exposes the probes on the microneedles topolynucleotide, peptide, and protein biomarkers within the cells. Theprobes on the microneedles can be allowed to hybridize 502 to thebiomarkers for a specified period of time at physiological conditions insitu, e.x., the probes can be allowed to hybridize for about 30 minutesat physiological body temperature (about 37° C.). Areverse-transcriptase-PCR (RT-PCR) assay 503 can be utilized to convertthe hybridized RNA into DNA. A standard PCR protocol 504 can be utilizedto amplify the product of 503.

FIG. 6 illustrates an overview of the method of providing a treatmentwith a device of the invention. 601 illustrates a clinician conducting avisual inspection of a subject's skin. A clinician 601 can determinethat a skin 602, or a portion of the skin, appears healthy or unhealthy.A clinician can touch the skin of the subject with a device of theinvention, thereby contacting a probe for a biomarker to the skin of thesubject in a non-invasive manner. 603 is a diagram of a surface of adevice that was described in greater detail in FIG. 5. 604 illustrates aPCR assay that is performed to amplify the hybridized biomarker. 605depicts a clinician returning the results of an analysis to a subject.

Example 5. In Vivo Testing of Modified Polymer Microneedle Arrays

This Example describes the in vivo detection of mouse actin using themodified polymer microneedle arrays described in this application. Thepolycarbonate microneedles were modified with a mouse actin ssDNA probe,and a sample of microneedles that had been coupled with a ssDNA probewas also coated with hyaluronidase. Mice (n=4; A/J, Swiss Webster) weretreated with the ssDNA-modified polycarbonate microneedles. Themicroneedle arrays were applied to the shaved rear leg of mice usingthumb pressure and held in place for approximately 10 seconds. mRNA thatwas bound to the arrays was then reverse transcribed into cDNA directlyon the microneedle surface by inverting the array over a glass slide,sealing the space between the needle base and slide with oil, andplacing the slide on a heat block for 30 minutes at 50° C. The reactionmixture was then transferred from the slide into a PCR tube andamplified using traditional PCR techniques. The cycling program went asfollows: 94° C. for 1 min; 40 cycles of: 94° C. for 15 s, 55° C. for 30s, 68° C. for 60 s; 68° C. for 5 min. The samples were visualized by gelimaging and quantitated using densitometry.

Significant increases in the amount of mRNA isolated from microneedlesthat contained probes was observed. The difference between samples thatcontained the ssDNA probe and unmodified microneedle arrays was˜2-3-fold while the difference between samples containing both ssDNAprobes and hyaluronidase and unmodified arrays was ˜8-fold. This showsthat not only do the microneedles extract the target from the skin, butthat interruption of the extracellular matrix can facilitate theextraction process, resulting in higher yields. A number of enzymes,including but not limited to serine proteases, thiol proteases, andMMPs, could be useful in this process. Further specific examples ofenzymes include but are not limited to papain, hyaluronidase,streptokinase, streptodornase, trypsin, chymotrypsin,alpha-chymotrypsin, alpha-amlyase, DNase, collagenase, and sutilain.

Example 6. Isolation of Target mRNA from Homogenized Human Skin

This Example describes the isolation of target mRNA from homogenizedhuman skin.

Excess human skin including both cancerous and benign tissue wasacquired from Scripps Clinic, which was excised during Mohs micrographicsurgery. The skin was homogenized and the total RNA extracted using thefollowing procedure. Skin was homogenized in RNAlater™ using a MPBiomedical FastPrep-24 and Lysing Matrix D beads (˜30-40 mg tissue/tube)on a setting of 6.0 for 25 s. Total RNA was then isolated from thishomogenate using a RNA extraction kit (Qiagen™) according to themanufacturer's instructions. DNA probes with sequences complimentary tohuman beta-actin were conjugated to gold coated stainless steelmicroneedles, placed into RNA solutions isolated from homogenized skinand incubated for periods of time at room temperature or at 37° C.Stainless steel strips were isolated from the solutions and the boundmRNA was reverse transcribed to cDNA, which was then amplified andanalyzed using qRT-PCR. As a result, only the microneedles that werecoupled to the beta-actin probe and subsequently amplified using theappropriate beta-actin probe yielded measureable amounts of target (FIG.8). In this example, non-specific DNA sequences were conjugated to themicroneedle surface; a BMP-4: Taqman probe was used against a non-actintarget; and a Actb-human-1: beta-actin sequence was conjugated to thesurface of a microneedle.

Example 7. Detection of mRNA from a Mouse Skin mRNA Library UsingMicroneedle Arrays

In these experiments, target mRNA was isolated from a mouse skin mRNAlibrary (Zyagen™). The total concentration of mRNA in the pool was 250μg/mL. Gold-coated stainless steel microneedle arrays onto which probessDNA had been coupled were then added to the mRNA library (5 μL in 20μL it water) and incubated at 37° C. for approximately 10 minutes.Arrays were then washed gently and briefly allowed to air dry. cDNAsynthesis then was performed by adding reverse transcriptase reactionmix (3 μL 10× reverse transcriptase buffer, 6 μL 25 mM MgCl₂, 3 μL 0.1 MDTT, 1.5 μL RNase OUT™, 13 μL DEPC water, 2 μL 10 mM dNTP mix) to tubescontaining microneedle arrays and incubating at 42° C. for 2 minutes,followed by the addition of 1.5 μL of SuperScriptII™ reversetranscriptase. The reactions were cycled through the following program:42° C. for 50 min, 70° C. for 15 min, and then on ice for approximately15 min. RNase H was then added to each reaction (1.5 μL) and thereactions were incubated at 37° C. for 20 min. This was then followed byTaqMan™ PCR amplification using 20×TaqMan™ assay primer (1.5 μL), and2×TaqMan™ gene expression mix (15 μL). Reactions had a total volume of30 μL. After four cycles, 5 μL of the solution was removed and used asthe template for TaqMan™ qRT-PCR (40 cycles) performed as per themanufacturer's instructions.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

All publications, databases, GenBank sequences, patents, and patentapplications cited in this specification are herein incorporated byreference as if each was specifically and individually indicated to beincorporated by reference.

1. A device for detecting or extracting one or more nucleic acidbiomarkers from an in situ tissue or biological sample of a subject,wherein the device comprises two or more microneedles and one or morepolynucleotide probes specific for the nucleic acid biomarkers, whereinthe polynucleotide probes are attached to the two or more microneedlesand a center-to-center distance between the two or more microneedles isgreater than 200 μm and less than 1000 μm.
 2. The device of claim 1,comprising a plurality of polynucleotide probes specific for a pluralityof different biomarkers, wherein each polynucleotide probe is attachedto a different microneedle.
 3. The device of claim 1, comprising onepolynucleotide probe for a specific biomarker, wherein thepolynucleotide probe is attached to the two or more microneedles.
 4. Thedevice of claim 1, wherein the microneedles are made of a polymer, ametal or a ceramic.
 5. The device of claim 1, wherein the polynucleotideprobes are polynucleotides complementary to the nucleic acid biomarkers.6. The device of claim 5, wherein the microneedles are polycarbonate,and the polynucleotide probes are attached to the microneedles via athiol/amino bifunctional linker.
 7. (canceled)
 8. The device of claim 1,wherein the polynucleotide probes are attached to the microneedles via alinker as a spacer or to stabilize orientation of the polynucleotideprobes.
 9. The device of claim 1, wherein the two or more microneedlesare formed on a substrate.
 10. The device of claim 1, further comprisingreagents for amplifying and identifying the nucleic acid biomarkerscaptured by the polynucleotide probes.
 11. A method for detecting one ormore biomarkers from an in situ tissue or biological sample in asubject, comprising (a) providing the device of claim 1; (b) contactingthe device of claim 1 with the in situ tissue or biological sample ofthe subject such that the nucleic acid biomarkers are bound to thepolynucleotide probes; and (c) detecting the nucleic acid biomarkersthat are bound to the polynucleotide probes.
 12. The method of claim 11,wherein the device comprises a plurality of polynucleotide probesspecific for a plurality of different biomarkers, and wherein eachpolynucleotide probe is attached to a different microneedle.
 13. Themethod of claim 11, wherein the device comprises one polynucleotideprobe specific for a biomarker, and wherein the polynucleotide probe isattached to the two or more microneedles.
 14. The method of claim 11,wherein the microneedles are made of a polymer, a metal or a ceramic.15. The method of claim 11, wherein the polynucleotide probes arepolynucleotides complementary to the nucleic acid biomarkers.
 16. Themethod of claim 15, wherein the nucleic acid biomarkers bound to thepolynucleotide probes on the microneedles are detected by PCR.
 17. Themethod of claim 15, wherein the microneedles are polycarbonate, and thepolynucleotide probes are attached to the microneedles via a linker. 18.(canceled)
 19. (canceled)
 20. The method of claim 11, wherein themicroneedles are formed on a substrate.
 21. The method of claim 11,wherein the tissue is the subject's blood stream, and the microneedledevice is contacted with the blood stream by piercing the skin of thesubject.
 22. (canceled)
 23. The method of claim 1, wherein the two ormore microneedles are solid.
 24. The method of claim 1, wherein thepolynucleotide probes comprise DNA.